CD40 coding sequences

ABSTRACT

A simple and highly efficient method for cloning cDNAs from mammalian expression libraries based on transient expression in mammalian host cells has been discovered. The present invention specifically provides the CD40 cDNA sequence.

This application is a divisional of co-pending U.S. patent applicationSer. No. 07/983,647, filed Dec. 1, 1992, which is a continuation-in-partof U.S. patent application Ser. No. 553,759, filed Jul. 13, 1990, nowabandoned; which is a continuation-in-in-part of U.S. Ser. No.07/498,809 filed Mar. 23, 1990, now abandoned; which is acontinuation-in-part of U.S. Ser. No. 379,076, filed Jul. 13, 1989, nowabandoned; which is a continuation-in-part of U.S. Ser. No. 160,416,filed Feb. 25, 1988, now abandoned. Each of these predecessorapplications and all references cited herein are incorporated byreference in their entirety.

BACKGROUND

A basic tool in the field of recombinant genetics is the conversion ofpoly(A)⁺ mRNA to double-stranded (ds) cDNA, which then can be insertedinto a cloning vector and expressed in an appropriate host cell.Molecular cloning methods for ds cDNA have been reviewed, for example,by Williams, "The Preparation and Screening of a cDNA Clone Bank," inWilliamson, ed., Genetic Engineering, Vol. 1, p. 2, Academic Press, NewYork (1981); Maniatis, "Recombinant DNA", in Prescott, ed., CellBiology, Academic Press, New York (1980); and Efstratiadis et al.,"Cloning of Double-Stranded DNA," in Stelo et al., Genetic Engineering,Vol. 1, p. 15, Plenum Press, New York (1979).

A substantial number of variables affect the successful cloning of aparticular gene and cDNA cloning strategy thus must be chosen with care.A method common to many cDNA cloning strategies involves theconstruction of a "cDNA library" which is a collection of cDNA clonesderived from the total poly(A)⁺ mRNA derived from a cell of the organismof interest.

A mammalian cell may contain up to 30,000 different mRNA sequences, andthe number of clones required to obtain low-abundance mRNAs, forexample, may be much greater. Methods of constructing genomic eukaryoticDNA libraries in different expression vectors, including bacteriophagelambda, cosmids, and viral vectors, are known. Some commonly usedmethods are described, for example, in Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher,Cold Spring Harbor, N.Y. (1982).

Once a genomic cDNA library has been constructed, it is necessary toisolate from the thousands of host cells the cell containing theparticular human gene of interest. Many different methods of isolatingtarget genes from cDNA libraries have been utilized, with varyingsuccess. These include, for example, the use of nucleic acid probes,which are labeled mRNA fragments having nucleic acid sequencescomplementary to the DNA sequence of the target gene. When this methodis applied to cDNA clones of abundant mRNAs in transformed bacterialhosts, colonies hybridizing strongly to the probe are likely to containthe target DNA sequences. The identity of the clone then may be proven,for example, by in situ hybridization/selection (Goldberg et al.,Methods Enzymol., 68:206 (1979)) hybrid-arrested translation (Patersonet al., Proceedings of the National Academy of Sciences, 74:4370(1977)), or direct DNA sequencing (Maxam and Gilbert, Proceedings of theNational Academy of Sciences, 74:560 (1977); Maat and Smith, NucleicAcids Res., 5:4537 (1978)).

Such methods, however, have major drawbacks when the object is to clonemRNAs of relatively low abundance from cDNA libraries. For example,using direct in situ colony hybridization, it is very difficult todetect clones containing cDNA complementary to mRNA species present inthe initial library population at less than one part in 200. As aresult, various methods for enriching mRNA in the total population (e.g.size fractionation, use of synthetic oligodeoxynucleotides, differentialhybridization, or immunopurification) have been developed and are oftenused when low abundance mRNAs are cloned. Such methods are described,for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual,supra.

Many functional eukaryotic proteins initially exist in the form ofprecursor molecules which contain leader or signal sequences at theirN-terminal ends. These leader sequences bind to the cell membrane anddraw the remainder of the protein through the lipid bilayer, after whichthe signal sequence is cleaved from the protein by a signal peptidaseenzyme. The protein thus functions only after secretion from the cells(for example, insulin, serum albumin, antibodies, and digestive tractenzymes), or after the proteins have been anchored to the outer surfaceof a cell membrane (for example, histocompatibility antigens).

The cell surface antigens characteristic of mammalian T lymphocytes areadditional examples of proteins that anchor to the cell surface. Inmammals, certain cells derived from bone marrow mature into lymphocytes,which are present in the lymphoid organs, including the thymus, spleen,lymph nodes, and lymphoid aggregates, and also circulate activelythrough the blood and lymph systems. Mature lymphocyte cells may bedivided into two populations: thymus-dependent (T) lymphocytes andthymus-independent (B) lymphocytes. T lymphocytes migrate to theinterior of the thymus, where they undergo differentiativeproliferation. During their differentiation process, they expresscharacteristic cell surface membrane alloantigens, including Thy-1, TLA,gv-1, Ly-1, Ly-2, Ly-3, and Ly-5. As they mature, T lymphocytes lose theTLA antigens and some of the Thy-1 antigens, and gain histocompatibilityantigens, acquiring the membrane conformation typical of therecirculating T lymphocytes. This is described, for example, by Mota,"Activity of Immune Cells," in Bier et al., eds., Fundamentals ofImmunology, 2d Ed., Springer-Verlag, Berlin, pp. 35-62 (1986).

T lymphocytes are involved indirectly in the formation of antibodies andtheir activities thus have required complex analysis of cell function,rather than simple antibody titer measurement. Partly due to this, theirimportance in development of immunologic competence was not recognizeduntil relatively recently. Mature T lymphocytes synthesize and expressan unique pattern of surface glycoprotein antigens which serve asmarkers for identification of different T lymphocyte subpopulations,including T helper cells, T suppressor cells, and T cytotoxic cells.Each of these subpopulations plays a very important role in regulatingthe immune system. (Mota, supra).

In humans, the functional and phenotypic heterogeneity of T lymphocytesis well accepted. Two major subpopulations are known: effector T cellsmediating cellular immunity; and regulator T cells containing helper andsuppressor T lymphocytes. These two subpopulations have been definedwith heteroantisera, autoantibodies, and monoclonal antibodies directedat cell surface antigens. For example, earlier in their development,human lymphoid cells in the thymus express an antigen designated T11which reacts strongly to a monoclonal antibody designated Cluster ofDifferentiation 2 (CD2), and react slightly with monoclonal antibody CD5to cell surface antigen T1. During maturation, these cells lose T11(CD2) and acquire three new antigens defined by monoclonal antibodiesCD4, CD8, and CD1. With further maturation, the thymocytes cease toexpress cell surface antigens reactive with monoclonal antibody CD1,express the T3 antigen reactive with monoclonal antibody CD3, and thensegregate into two subpopulations which express either T4 (CD4) or T8(CD8) antigen. Immunologic competence is acquired at this stage, but isnot completely developed until thymic lymphocytes migrate outside thethymus. (Mota, supra.) In contrast with the majority of thymocytes,circulating T lymphocytes express the T1 (CD5) and T3 (CD3) antigens.The T4 (CD4) antigen is present on approximately 55-65% of peripheral Tlymphocytes, whereas the T8 (CD8) antigen is expressed on 20-30%. Thesetwo subpopulations correspond to helper and to suppressor and cytotoxicT cells, respectively.

In addition to providing a convenient means of distinguishing Tlymphocyte subpopulations, these cell surface antigens are important formature T cell activation and effector function. T cell activationinvolves a complex series of cell surface interactions between the Tcell and the target cell or stimulator cell in addition to binding ofthe T cell receptor to its specific antigen.

For example, CD2, the human T cell erythrocyte receptor, allowsthymocytes and T-lymphocytes to adhere to target cells (e.g.,erythrocytes) and to thymic epithelium. This occurs via a specificmolecular ligand for CD2, designated LFA-3, in humans, which is a widelydistributed surface antigen. This phenomenon has long been employed todetect, assay and purify human cells producing antibodies to sheeperythrocytes and serves as the basis for the E-rosette test, firstdescribed by Zaalberg, Nature 202:1231 (1964). CD2LFA-3 interactionsalso have been shown to mediate cytolytic target conjugation (Shaw etal., Nature 323:262-264 (1986), and the mixed lymphocyte reaction(Martin et al., J. Immunol. 131:180-185 (1983). Anti-CD2 monoclonalantibodies can directly activate peripheral T-lymphocytes via anantigen-independent pathway (Meuer et al., Cell 36:897-906 (1984)),indicating an even wider immunoregulatory role for CD2.

Recognition that T lymphocytes are the main effectors of cell-mediatedimmunity and also are involved as helper or suppressor cells inmodulating the immune response has resulted in a significantcontribution to the increasing practical application of clinicalimmunology to medicine. The scope of this application includes defenseagainst infections, prevention of diseases by immunization, organtransplantation, blood banking, and treatment of deficiencies of theimmune system and a variety of disorders that are mediated byimmunologic mechanisms. Moreover, immunologic techniques frequently areused in the clinical laboratory, as in the measurement of hormones anddrugs. Clinical immunology is described, for example, in Weir, ed.,Handbook of Experimental Immunology in Four Volumes: Volume 4:Applications of Immunological Methods in Biomedical Sciences, 4th Ed.,Blackwell Scientific Publications, Oxford (1986); Boguslaski et al.,eds., Clinical Immunochemistry: Principles of Methods and Applications,Little, Brown & Co., Boston (1984); Holborow et al., eds., Immunology inMedicine: A Comprehensive Guide to Clinical Immunology, 2d Ed., Grune &Stratton, London (1983); and Petersdorf et al., eds., Harrison'sPrinciples of Internal Medicine, 10th ed., McGraw-Hill, New York,publisher, pp. 344-391 (1983). Clearly, a more thorough understanding ofthe proteins which mediate the immune system would be of significantvalue in clinical immunology.

Use of mammalian expression libraries to isolate cDNAs encodingmammalian proteins such as those described above would offer severaladvantages. For example, the protein expressed in a mammalian host cellshould be functional and should undergo any normal posttranslationalmodification. A protein ordinarily transported through the intracellularmembrane system to the cell surface should undergo the completetransport process. A mammalian expression system also would allow thestudy of intracellular transport mechanisms and of the mechanism thatinsert and anchor cell surface proteins to membranes.

One common mammalian host cell, called a "COS" cell, is formed byinfecting monkey kidney cells with a mutant viral vector, designatedsimian virus strain 40 (SV40), which has functional early and lategenes, but lacks a functional origin of replication. In COS cells, anyforeign DNA cloned on a vector containing the SV40 origin of replicationwill replicate because SV40 T antigen is present in COS cells. Theforeign DNA will replicate transiently, independently of the cellularDNA.

With the exception of some recent lymphokine cDNAs isolated byexpression in COS cells (Wong, G. G., et al., Science 228:810-815(1985); Lee, F. et al., Proceedings of the National Academy of Sciences,USA 83:2061-2065 (1986); Yokota, T., et al., Proceedings of the NationalAcademy of Sciences, USA 83:5894-5898 (1986); Yang, Y., et al., Cell47:3-10 (1986)), however, few cDNAs in general are isolated frommammalian expression libraries. There appear to be two principal reasonsfor this: First, the existing technology (Okayama, H. et al., Mol. Cell.Biol. 2:161-170 (1982)) for construction of large plasmid libraries isdifficult to master, and library size rarely approaches that accessibleby phage cloning techniques. (Huynh, T. et al., In: DNA Cloning Vol. I,A Practical Approach, Glover, D. M. (ed.), IRL Press, Oxford (1985), pp.49-78). Second, the existing vectors are, with one exception (Wong, G.G., et al., Science 228:810-815 (1985)), poorly adapted for high levelexpression, particularly in COS cells. The reported successes withlymphokine cDNAs do not imply a general fitness of the methods used,since these cDNAs are particularly easy to isolate from expressionlibraries. Lymphokine bioassays are very sensitive ((Wong, G. G., etal., Science 228:810-815 (1985); Lee, F. et al., Proceedings of theNational Academy of Sciences, USA 83:2061-2065 (1986); Yokota, T. etal., Proceedings of the National Academy of Sciences, USA 83:5894-5898(1986); Yang, Y. et al., Cell 47:3-10 (1986)) and the mRNAs aretypically both abundant and short (Wong, G. G. et al., Science228:810-815 (1985); Lee, F., et al., Proceedings of the National Academyof Sciences, USA 83:2061-2065 (1986); Yokota, T., et al., Proceedings ofthe National Academy of Sciences, USA 83:5894-5898 (1986); Yang, Y., etal., Cell 47:3-10 (1986)).

Thus, expression in mammalian hosts previously has been most frequentlyemployed solely as a means of verifying the identity of the proteinencoded by a gene isolated by more traditional cloning methods. Forexample, Stuve et al., J. Virol. 61(2):327-335 (1987), cloned the genefor glycoprotein gB2 of herpes simplex type II strain 333 by plaquehybridization of M13-based recombinant phage vectors used to transformcompetent E. coli JM101. The identity of the protein encoded by theclone thus isolated was verified by transfection of mammalian COS andChinese hamster ovary (CHO) cells. Expression was demonstrated byimmunofluorescence and radioimmunoprecipitation.

Oshima et al. used plaque hybridization to screen a phage lambda gt11cDNA library for the gene encoding human placental beta-glucuronidase.Oshima et al., Proceedings of the National Academy of Sciences, U.S.A.84:685-689 (1987). The identity of isolated cDNA clones was verified byimmunoprecipitation of the protein expressed by COS-7 cells transfectedwith cloned inserts using the SV40 late promoter.

Transient expression in mammalian cells has been employed as a means ofconfirming the identity of genes previously isolated by other screeningmethods. Gerald et al., Journal of General Virology 67:2695-2703(1986).Mackenzie, Journal of Biological Chemistry 261:14112-14117 (1986); Seifet al., Gene 43:1111-1121 (1986); Orkin et al., Molecular and CellularBiology 5(4):762-767 (1985). These methods often are inefficient andtedious and require multiple rounds of screening to identify full-lengthor overlapping clones. Prior screening methods based upon expression offusion proteins are inefficient and require large quantities ofmonoclonal antibodies. Such drawbacks are compounded by use ofinefficient expression vectors, which result in protein expressionlevels that are inadequate to enable efficient selection.

SUMMARY OF THE INVENTION

The present invention relates to a powerful new method for cloning cDNAencoding cell surface antigens, to a method of constructing cDNAlibraries, to high efficiency expression vectors particularly suited forhigh level expression in eukaryotic host cells, and to the isolatednucleotide sequences and their encoded products.

The highly efficient cloning technique of the present invention is basedupon transient expression of antigen in eukaryotic cells and physicalselection of cells expressing the antigen by adhesion to anantibody-coated substrate, such as a culture dish. The methods of thepresent invention are useful for the isolation and molecular cloning ofany protein which can be expressed and transported to the cell surfacemembrane of a eukaryotic cell.

The method for cloning cDNA encoding a cell surface antigen of thepresent invention comprises preparing a cDNA library; introducing thiscDNA library into eukaryotic mammalian cells, preferably tissue culturecells; culturing these cells under conditions allowing expression of thecell surface antigen; exposing the cells to a first antibody orantibodies directed against the cell surface antigen, thereby allowingthe formation of a cell surface antigen-first antibody complex;subsequently exposing the cells to a substrate coated with a secondantibody directed against the first antibody, thereby causing cellsexpressing the cell surface antigen to adhere to the substrate via theformation of a cell surface antigen-first antibody-second antibodycomplex; and separating adherent from non-adherent cells.

By means of the cloning method of the present invention, isolation andmolecular cloning of genes encoding such cell surface antigens as thefollowing have been accomplished: the CD1a, CD1b, CD1c, CD2, CD6, CD7,CD13, CD14, CD16, CD19, CD20, CD22, CD26, CD27, CD28, CD31, CDw32a,CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD53,ICAM, LFA-3, FcRIa, FcRIb, TLiSa, and Leu8 antigens. The nucleotidesequences of genes cloned by the method of the present invention havebeen determined and the amino acid sequences of the encoded proteinshave been identified. A cloned gene, such as that encoding CD1a, CD1b,CD1c, CD2, CD6, CD7, CD13, CD14, CD16, CD19, CD20, CD22, CD26, CD27,CD28, CD31, CDw32a, CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40,CD43, CD44, CD53, ICAM, LFA-3, FcRIa, FcRIb, TLiSa, and Leu8, is alsothe subject of the present invention.

Once the gene encoding an antigen has been cloned according to themethod of the present invention, that gene can be expressed in aprokaryotic or a eukaryotic host cell to produce the encoded protein orportion thereof in substantially pure form such as it does not exist innature. Another aspect of the present invention relates to substantiallypure cell surface antigens, particularly: CD1a, CD1b, CD1c, CD2, CD6,CD7, CD13, CD14, CD16, CD19, CD20, CD22, CD26, CD27, CD28, CD31, CDw32a,CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD53,ICAM, LFA-3, FcRIa, FcRIb, TLiSa, and Leu8 antigens and their functionalanalogues and equivalents. The primary amino acid sequences of the CD1a,CD1b, CD2, CD7, CD14, CD16, CD19, CD20, CD22, CD27, CD28, CDw32a,CDw32b, CD33, CD34, CD40, CD44, CD53, ICAM, LFA-3, FcRIa, FcRIb, TLiSaand Leu8 antigens have been determined. The invention thus also relatesto the amino acid sequences of those antigens and their functionalequivalents and to the nucleotide sequences encoding those antigens.

This invention also relates to high efficiency cDNA expression vectorswhich allow the generation of very large mammalian expression librariesand yield large amounts of protein in mammalian host cells, resulting inefficient selection. In a particular embodiment of this invention, acDNA expression vector comprises a suppressor tRNA gene; an SV40 origin;a synthetic transcription unit, comprising a chimeric promoter composedof human cytomegalovirus AD169 immediate early enhancer sequence fusedto the HIV LTR -60 to +80 sequences, inserted between the suppressortRNA gene and the SV40 origin; a polylinker comprising two BstXI sitesseparated by a replaceable DNA sequence and flanked by XbaI sites; andan SV40 small t antigen splice and early region polyadenylation signals.

A further aspect of the present invention comprises a synthetictranscription unit for use in a cDNA expression vector, comprising achimeric promoter composed of human cytomegalovirus AD169 immediateearly enhancer sequences fused to HIV LTR -60 to +80 sequences. Thesmall size and particular arrangement of the sequences of the cDNAexpression vector of the present invention allow highly efficientreplication in host mammalian tissue culture cells, such as COS cells.Moreover, this vector employs a polylinker containing two inverted BstXIsites separated by a short replaceable DNA segment, which allows the useof very efficient oligonucleotide-based cDNA insertion strategy.

In another aspect, the present invention comprises a vector comprisingtwo identical BstXI sites in inverted orientation each with respect tothe other, which BstXI sites are separated by a short replaceable DNAfragment. Another aspect of the invention is a polylinker as describedabove.

A further aspect of the invention relates to an oligonucleotide-basedcDNA insertion method, comprising ligating synthetic DNAoligonucleotides to the cDNA segment desired to be inserted into avector, the synthetic DNA oligonucleotides giving the same terminalsequences as those of the short replaceable DNA fragment of thepolylinker of the invention, and inserting the resulting cDNA segmentplus synthetic DNA oligonucleotide terminal sequences into thepolylinker of the vector, from which the short replaceable DNA fragmentpreviously has been removed.

In preparing cDNA libraries according to the present invention, it hasbeen discovered that many tumors are heavily infiltrated by macrophagesand lymphocytes, and thus may be employed as a source of macrophage orlymphocyte transcripts to good effect, instead of tumor cell linescommonly used. In another aspect, then, the present invention relates tothe use of tumor cells, particularly human tumor cells, to prepare cDNAlibraries for use according to the methods of the present invention.

Another advantage of the powerful selection system of the presentinvention is that directional insertion of the cDNA is not necessary.The method of the present invention results in library constructionefficiencies which are on a par with those described for phage vectorssuch as lambda gt10 and lambda gt11, with the additional advantage thatclones generated according to the methods of the present invention areeasier to manipulate.

The immunoselection technique of the present invention allows efficientuse of antibodies, which may be monoclonal or polyclonal, in relativelysmall absolute amounts. The method of the present invention also isquite rapid. Generally, three or fewer cycles of immunoselection andrescue are required to isolate a target cDNA clone. Thus, the method ofthe present invention also results in the efficient use of labor andmaterials when cloning genes encoding cell surface antigens. Asdescribed above, this method has been employed to successfully clonegenes encoding cell surface antigens associated with mammalian Tlymphocytes (e.g. antigens CD1a, CD1b, CD1c, CD2, CD6, CD7, CD13, CD14,CD16, CD19, CD20, CD22, CD26, CD27, CD28, CD31, CDw32a, CDw32b, CD33,CD34, CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD53, ICAM, LFA-3,FcRIa, FcRIb, TLiSa, and Leu8).

The purified genes and proteins of the present invention are useful forimmunodiagnostic and immunotherapeutic applications, including thediagnosis and treatment of immune-mediated infections, diseases, anddisorders in animals, including humans. They can also be used toidentify, isolate and purify other antibodies and antigens. Suchdiagnostic and therapeutic uses comprise yet another aspect of thepresent invention. Moreover, the substantially pure proteins of thepresent invention may be prepared as medicaments or pharmaceuticalcompositions for therapeutic administration. The present inventionfurther relates to such medicaments and compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B.

Nucleotide sequence of expression vector piH3

Nucleotides 1-589 are derived from pMB1 origin (pBR322 ori); nucleotides590-597 are derived from the SacII linker (ACCGCGT); nucleotides 598-799are derived from the synthetic tyrosine suppressor tRNA gene (supFgene); nucleotides 800-947 are derived from a remnant of the ASV LTRfragment (PvuII to MIu1); nucleotides 948-1500 are derived from thehuman cytomegalovirus AD169 enhancer; nucleotides 1501-1650 are derivedfrom HIV TATA and tat-responsive elements; nucleotides 1651-1716 arederived from the piLNXAN polylinker (HindIII to Xba); nucleotides1717-2569 are derived from pSV to splice and poly-Addition signals;nucleotides 2570-2917 are derived from the SV40 origin of replication(PvuII to (HindIII); and nucleotides 2918-2922 are derived from piVX,remnant of R1 site from polylinker.

FIGS. 2A-2B.

Nucleotide sequence of the CD2cDNA insert

Nucleotide numbering is given in parentheses at right, amino acidnumbering, left. Locations of the potential sites for addition ofasparagine-linked carbohydrate (CHO) are shown, as well as the predictedtransmembrane (TM) sequence. The amino acid sequence is numbered fromthe projected cleavage site of the secretory signal sequence.

FIG. 3.

Restriction map of the CDM8 expression vector

The CDM8 vector includes a deleted version of a mutant polyoma virusearly region selected for high efficiency expression in both murine andmonkey cells. Substantially all of the human immunodeficiency promoterregion has been replaced with the cognate sequences of the humancytomegalovirus immediate early promoter, and by inclusion of abacteriophage T7 promoter between the eukaryotic promoter and the siteof cDNA insertion. Arrows indicate the direction of transcription.

FIGS. 4A-4B.

Nucleotide sequence and corresponding amino acid sequence of the LFA-3antigen

WOP cells transfected with a clone encoding the LFA-3 antigen weredetected by indirect immunofluorescence, amplified and sequenced. FIG.4A shows the 874 base pair insert containing an open reading frame of237 residues originating at a methionine codon, and terminating in aseries of hydrophobic residues. Hydrophobic and hydrophilic regionswithin this open reading frame are shown in FIG. 4B.

FIG. 5.

Restriction Map of the piH3M vector

The direction of transcription is indicated by an arrow. Restrictionendonuclease sites flanking the BstXI cloning sites are shown.

FIGS. 6A-6D.

Nucleotide sequence of the piH3M vector

There are 7 segments. Residues 1-587 are from the pBR322 origin ofreplication, 588-1182 from the M13 origin, 1183-1384 from the supF gene,1385-2238 are from the chimeric cytomegalovirus/human immunodeficiencyvirus promoter, 2239-2647 are from the replaceable fragment, 2648-3547from plasmid pSV2 (splice and polyadenylation signals), and 3548-3900from the SV40 virus origin.

FIGS. 7A-7B.

Nucleotide sequence of the CD28 cDNA

Nucleotide numbering is given in parentheses at right, amino acidnumbering, center and left. Location of the potential sites for additionof asparagine-linked carbohydrate (CHO) are shown, as well as thepredicted transmembrane (TM) sequence. The amino acid sequence isnumbered from the projected cleavage site of the secretory signalsequence.

FIGS. 8A-8B.

Nucleotide sequence of the CD7 cDNA insert

Nucleotide numbering is given in parentheses at right. Splice donor andacceptor sites indicated by (/). The location of the potential sites foraddition of asparagine-linked carbohydrate (CHO) are shown, thepotential fatty acid esterification site is denoted (*), and thepredicted transmembrane domain (TM) is underlined. Nucleotide sequencespotentially involved in hairpin formation are denoted by (.). Thepresumed polyadenylation signal is underlined.

FIGS. 9A-9B.

Nucleotide sequence of the CDw32 cDNA

Nucleotide number is given in the parenthesis at right, amino acidnumbering, center and left. Locations of the potential sites foraddition of asparagine-linked carbohydrate (CHO) are shown, as well asthe predicted transmembrane (TM) sequence. The amino acid sequence isnumbered from the projected cleavage site of the secretory signalsequence. Cysteine residues are underscored with asterisks.

FIGS. 10A-10B.

Sequence of the CD20.4 cDNA

The sites of potential N-linked glycosylation are denoted by the symbol--CHO--; the hydrophobic regions are underscored. The site of thepoly(A)⁺ tail in clone CD20.6 is denoted by an asterisk.

FIG. 10C.

Hydrophobicity profile of the amino acid sequence in A.

FIGS. 11A-11C.

Sequence of ICAM-1

Complete nucleotide sequence of ICAM-1 cDNA insert and predicted proteinsequence. Nucleotide numbering is at left, amino acid numbering, center.The RGE motif at position 128 is underlined, the potential N-linkedglycosylation sites are indicated by --CHO-- and the transmembranedomain by -TM-. The amino acid sequence is numbered from the projectedcleavage site of the signal peptide. Sequencing was by dideoxy-chaintermination (Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74:5463-5467(1977)), using a combination of subclones, and specificoligonucleotides.

FIGS. 12A-12B.

Nucleotide sequence of CD19

FIGS. 13A-13B.

Nucleotide sequence of CD20

FIGS. 14A-14B.

Nucleotide sequence of CDw32a

FIGS. 15A-15B.

Nucleotide sequence of CDw32b

FIG. 16.

Nucleotide sequence of CD40

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a novel method for cloning cDNA encoding acell surface antigen and to a method of constructing cDNA libraries. Italso relates to particular cDNA expression vectors and componentsthereof, nucleotide sequences or genes isolated by the method,substantially pure cell surface antigens encoded by the cDNA segments,and methods of using the isolated nucleotide sequences and encodedproducts.

In the following description, reference will be made to variousmethodologies known to those of skill in the art of recombinantgenetics. Publications and other materials setting forth such knownmethodologies to which reference is made are incorporated herein byreference in their entireties. Standard reference works setting forththe general principles of recombinant DNA technology include Darnell, J.E. et al., Molecular Cell Biology, Scientific American Books, Inc.,publisher, New York, N.Y. (1986); Lewin, B. M., Genes II, John Wiley &Sons, publisher, New York, N.Y. (1985); Old, R. W. et al., Principles ofGene Manipulation: An Introduction to Genetic Engineering, 2d edition,University of California Press, Berkeley, Calif. (1981); and Maniatis,T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y. (1982).

By "cloning" is meant the use of in vitro recombination techniques toinsert a particular gene or other DNA sequence into a vector molecule.In order to successfully clone a desired gene, it is necessary to employmethods for generating DNA fragments, for joining the fragments tovector molecules, for introducing the composite DNA molecule into a hostcell in which it can replicate, and for selecting the clone having thetarget gene from amongst the recipient host cells.

By "cDNA" is meant complementary or copy DNA produced from an RNAtemplate by the action of RNA-dependent DNA polymerase (reversetranscriptase). Thus a "cDNA clone" means a duplex DNA sequencecomplementary to an RNA molecule of interest, carried in a cloningvector.

By "cDNA library" is meant a collection of recombinant DNA moleculescontaining cDNA inserts which together comprise the entire genome of anorganism. Such a cDNA library may be prepared by art-recognized methodsdescribed, for example, in Maniatis et al., Molecular Cloning: ALaboratory Manual, supra. Generally, RNA is first isolated from thecells of an organism from whose genome it is desired to clone aparticular gene. Preferred for the purposes of the present invention aremammalian, and particularly human, cell lines. More preferred are thehuman tumor cell line HPB-ALL and the human lymphoblastoid cell line JY.Alternatively, RNA can be isolated from a tumor cell, derived from ananimal tumor, and preferably from a human tumor. Thus, a library may beprepared from, for example, a human adrenal tumor, but any tumor may beused.

The immunoselection cloning method of the present invention comprisesthe preparation of a cDNA library by extracting total RNA including aparticular gene from a cell, synthesizing a series of complementarydouble-stranded cDNA fragments from the RNA and introducing these cDNAfragments into mammalian cells in tissue culture. The mammalian cellsare maintained under conditions which allow them to express the protein(i.e. the cell surface antigen). The resulting cells are exposed to afirst antibody or pool (group) of antibodies directed against the cellsurface antigen. This results in formation of a cell surfaceantigen-first antibody complex. The complexes are exposed to a substrateto which is coated or bound a second antibody directed against the firstantibody. Cells expressing the cell surface antigen adhere to thesubstrate (because of formation of a cell surface antigen-firstantibody-second antibody complex). Adherent cells are separated fromnonadherent cells.

Isolation of total RNA

The guanidium thiocyanate/CsCl method of isolating total RNA ispreferred. More preferred is a guanidium thiocyanate/LiCl variant of theGuSCN/CsCl method, which has added capacity and speed. Briefly, for eachml of mix desired, 0.5 g GuSCN are dissolved in 0.58 ml of 25% LiCl(stock filtered through 0.45 micron filter) and 20 μl of mercaptoethanolis added. Cells are spun out and the pellet is dispersed on walls byflicking, add 1 ml of Solution up to 5×10⁷ cells. The resultingcombination is sheared by polytron until nonviscous. For small scalepreps (less than 10⁸ cells) layer 2 ml of sheared mix on 1.5 ml of 5.7MCsCl (RNase free; 1.26 g CsCl added to every ml 10 mM EDTA pH 8),overlay with RNase-free water and spin SW55 50 k rpm 2 h. For largescale preps, layer 25 ml on 12 ml CsCl in a SW28 tube, overlay, and spin24 k rpm 8 h. Aspirate contents carefully with a sterile pasteur pipetconnected to a vacuum flask. Once past the CsCl interface, scratch aband around the tube with the pipet tip to prevent the layer on the wallof the tube from creeping down. The remaining CsCl solution isaspirated. The pellets are taken up in water (do not try to redissolve).1/10 vol. NaOAc and 3 vol. EtOH are added and the resulting combinationis spun. If necessary, the pellet is resuspended in water (e.g., at70°). Adjust concentration to 1 mg/ml and freeze. Small RNA (e.g. 5 S)does not come down. For small amounts of cells, scale down volumes andoverlay GuSCN with RNase-free water on gradient (precipitation isinefficient when RNA is dilute).

Preparation of poly A⁺ RNA

Next, polyA⁺ RNA may be prepared, preferably by the oligo dT selectionmethod. Briefly, a disposable polypropylene column is prepared bywashing with 5M NaOH and then rinsing with RNase-free water. For eachmilligram total RNA about 0.3 ml (final packed bed) oligo dT celluloseis used. Oligo dT cellulose is prepared by resuspending about 0.5 ml ofdry powder in 1 ml of 0.1M NaOH and transferring it into the column, orby percolating 0.1 NaOH through a previously used column (columns can bereused many times). This is washed with several column volumes ofRNase-free water, until pH is neutral, and rinsed with 2-3 ml of loadingbuffer. The column bed is then removed into a sterile 15 ml tube using4-6 ml of loading buffer. The total RNA is heated to 70° C. for 2-3min., LiCl from RNase-free stock is added (to 0.5M), and combined witholigo dT cellulose in a 15 ml tube. This is followed by vortexing oragitation for 10 min. The result is poured into a column and washed with3 ml loading buffer and then 3 ml of middle wash buffer. mRNA is eluteddirectly into an SW55 tube with 1.5 ml of 2 mM EDTA, 0.1% SDS; the firsttwo or three drops are discarded.

Eluted mRNA is precipitated by adding 1/10 vol. 3M NaOAc and filling thetube with EtOH. This is then mixed, chilled for 30 minutes at -20° C.,and spun at 50 k rpm at 5° C. for 30 min. The EtOH is poured off and thetube is air dried. The mRNA pellet is resuspended in 50-100 μl ofRNase-free water. Approximately 5 μl is melted at 70° C. inMOPS/EDTA/formaldehyde and run on an RNase-free 1 % agarose gel to checkquality.

cDNA Synthesis

From this, cDNA is synthesized. A preferred method of cDNA synthesis isa variant of that described by Gubler and Hoffman (Gene, 25:263-269(1982)). This is carried out as follows:

a. First Strand. 4 μg of mRNA and heated to about 100° C. in a microfugetube for 30 seconds and quenched on ice. The volume is adjusted to 70 μlwith RNase-free water. The following are added: 20 μl of RT1 buffer, 2μl of RNAse inhibitor (Boehringer 36 U/μl), 1 ul of 5 μg/μl of oligo dT(Collaborative Research), 2.5 μl of 20 mM dXTP's (ultrapure), 1 μl of 1MDTT and 4 l of RT-LX (Life Science, 24 U/μl). The resulting combinationis incubated at 42° C. for 40 min. It is heated to inactivate (70° C. 10min).

b. Second Strand. 320 μl of RNAse free water, 80 μl of RT2 buffer, 5 μlof DNA Polymerase I (Boehringer, 5 U/μl), 2 μl RNAse H (BRL 2 U/μl).Incubate at 15° C. for 1 hr and 22° C. for 1 hr. Add 20 μl of 0.5M EDTApH 8.0, phenol extract and EtOH precipitate by adding NaCl to 0.5M,linear polyacrylamide (carrier) to 20 μg/ml, and filling tube with EtOH.Spin 2-3 minutes in microfuge, remove, vortex to dislodge precipitatehigh up on wall of tube, and respin 1 minute.

c. Adaptors. Resuspend precipitated cDNA in 240 μl of TE (10/1). Add 30μl of 10× low salt buffer, 30 μl of 10× low salt buffer, 30 μl of 10×ligation additions, 3 μl (2.4 μg) of kinased 12-mer adaptor, 2 μl (1.6μg) of kinased 8-mer adaptor, and 1 μl of T4 DNA ligase (BioLabs, 400U/μl, or Boehringer, 1 Weiss unit/ml). Incubate at 15° C. overnight.Phenol extract and EtOH precipitate as above (no extra carrier nowneeded), and resuspend in 100 μl of TE.

Use of cDNA fragments in expression vectors

For use with the BstXI-based cDNA expression vectors of the invention(see infra), oligonucleotide segments containing terminal sequencescorresponding to BstXI sites on the vectors are ligated to the cDNAfragment desired to be inserted. The resulting fragments are pooled byfractionation. A preferred method is as follows:

Prepare a 20% KOA, 2 mM EDTA, 1 μg/ml EthBr solution and a 5% KOAc, 2 mMEDTA, 1 μg/ml EthBr solution. Add 2.6 ml of 20% KOAc solution to backchamber of a small gradient maker. Remove air bubble from tubeconnecting the two chambers by allowing solution to flow into the frontchamber and then tilt back. Close passage between chambers, and add 2.5ml. of the 5% solution to the front chamber. If there is liquid in thetubing from a previous run, allow the 5% solution to run just to the endof the tubing, and then return to chamber. Place the apparatus on astirplate, set the stir bar moving as fast as possible, open thestopcock connecting the two chambers and then open the front stopcock.Fill a polyallomer SW55 tube from the bottom with the KOAc solution.Overlay the gradient with 100 μl of cDNA solution. Prepare a balancetube and spin the gradient for 3 hrs at 50 k rpm at 22° C. To collectfractions from the gradient, pierce the SW55 tube with a butterflyinfusion set (with the luer hub clipped off) close to the bottom of thetube and collect three 0.5 ml fractions and then 6 0.25 ml fractionsinto microfuge tubes (about 22 and 11 drops respectively). EtOHprecipitate the fractions by adding linear polyacrylamide to 20 μg/mland filling the tube to the top with EtOH. After cooling tubes, spinthem in a microfuge for 3 min. Vortex and respin 1 min. Rinse pelletswith 70% EtOH (respin). Do not dry to completion. Resuspend each 0.25 mlfraction in 10 μl of TE. Run 1 μl on a 1% agarose minigel. Pool thefirst three fractions, and those of the last six which contain nomaterial smaller than 1 kb.

Suppressor tRNA plasmids may be propagated by known methods. In apreferred method according to the present invention, supF plasmids canbe selected in nonsuppressing hosts containing a second plasmid, p3,which contains amber mutated ampicillin and tetracycline drug resistanceelements (Seed, 1983). The p3 plasmid is derived from PR1, is 57 kb inlength, and is a stably maintained, single copy episome. The ampicillinresistance of this plasmid reverts at a high rate, so that amp^(r)plasmids usually cannot be used in p3-containing strains. Selection fortet resistance alone is almost as good as selection for amp+tetresistance. However, spontaneous appearance of chromosomal suppressortRNA mutations presents an unavoidable background (frequency about 10⁻⁹)in this system. Colonies arising from spontaneous suppressor mutationsare usually bigger than colonies arising from plasmid transformation.Suppressor plasmids typically are selected for in LB medium containingamp at 12.5 μg/ml and tet at 7.5 μg/ml. For large plasmid preps, M9casamino acids medium containing glycerol (0.8%) may be used as a carbonsource, and the bacteria grown to saturation.

Vector DNA may be isolated by known methods. The following method ispreferred for plasmid from 1 liter of saturated cells:

Spin down cells in 1 liter J6 bottles, 4.2 k rpm, 25 minutes. Resuspendin 40 ml 10 mM EDTA pH 8 (Thump on soft surface). Add 80 ml 0.2M NaOH,1% SDS, swirl until clearish, viscous. Add 40 ml 5M KOAc, pH 4.7 (2.5MKOAc, 2.5M HOAc) shake semi-vigorously (until lumps are 2-3 mm in size).Spin (same bottle) 4.2K rpm, 5 min. Pour supernatant through cheeseclothinto 250 ml bottle. Fill bottle with isopropyl alcohol. Spin J6, 4.2 krpm, 5 min. Drain bottle, rinse gently with 70% EtOH (avoid fragmentingthe pellet). Invert bottle, and remove traces of EtOH with Kimwipe.Resuspend in 3.5 ml Tris base/EDTA 20 mM/10 mM. Add 3.75 ml ofresuspended pellet to 4.5 g CsCl. Add 0.75 ml 10 mg/ml ethidium bromide,mix. Fill VTi80 tubes with solution. Run at a speed of 80 rpm for 2.5hours or longer. Extract bands by visible light with 1 ml syringe and 20gauge or lower needle. Cut top off tube, insert the needle upwards intothe tube at an angle of about 30° with respect to the tube, (i.e., asshallowly possible) at a position about 3 mm beneath the band, with thebevel of the needle up. After the band is removed, pour tube contentsinto bleach. Deposit extracted bands in 13 ml Sarstedt tube. Fill tubeto top with n-butanol saturated with 1M NaCl, extract. If a very largequantity of DNA is obtained, reextract. Aspirate butanol into trapcontaining 5M NaOH (to destroy ethidium). Add about equal volume 1Mammonium acetate to DNA (squirt bottle). Add about 2 volumes 95% ethanol(squirt bottle). Spin 10K rpm, 5 min. J2-21. Rinse pellet carefully with70% ethanol. Dry with swab, or lyophilizer.

The vector may be prepared for cloning by known methods. A preferredmethod begins with cutting 20 μg of vector in a 200 μl reaction with 100units of BstXI (New York Biolabs), cutting at 50° C. overnight in awell-thermostatted water bath (i.e., circulating water bath). Prepare 2KOAc 5-20% gradients in SW55 tubes as described above. Add 100 μl of thedigested vector to each tube and run for 3 hrs, 50K rpm at 22° C.Examine the tube under 300 nm UV light. The desired band will havemigrated 2/3 of the length of the tube. Forward trailing of the bandmeans the gradient is overloaded. Remove the band with a 1 ml syringeand 20 gauge needle. Add linear polyacrylamide and precipitate theplasmid by adding 3 volumes of EtOH. Resuspend in 50 μl of TE. Set upligations using a constant amount of vector and increasing amounts ofcDNAs. On the basis of these trial ligations, set up large scaleligation, which can be accomplished by known methods. Usually the entirecDNA prep requires 1-2 μg of cut vector.

Adaptors may be prepared by known methods, but it is preferred toresuspend crude adaptors at a concentration of 1 μg/μl, add MgSO₄ to 10mM, and precipitate by adding 5 volumes of EtOH. Rinse with 70% EtOH andresuspend in TE at a concentration of 1 μg/μl. To kinase take 25 μl ofresuspended adaptors, add 3 μl of 10× kinasing buffer and 20 units ofkinase; incubate 37° C. overnight.

Preparation of buffers mentioned in the above description of preferredmethods according to the present invention will be evident to those ofskill. For convenience, preferred buffer compositions are as follows:

Loading Buffer: 0.5M LiCl, 10 mM Tris pH 7.5, 1 mM EDTA 0.1% SDS.

Middle Wash Buffer: 0.15M LiCl, 10 mM Tris pH 7.5, 1 mM EDTA 0.1% SDS.

Rt1 Buffer: 0.25M Tris pH 8.8 (8.2 at 42°), 0.25M KCl, 30 mM MgCl₂.

RT2 Buffer: 0.1M Tris pH 7.5, 25 mM MgCl₂, 0.5M KCl, 0.25 mg/ml BSA, 50mM DTT.

10× Low Salt: 60 mM Tris pH 7.5, 60 mM MgCl₂, 50 mM NaCl, 2.5 mg/ml BSA,70 mM Me.

10× Ligation Additions: 1 mM ATP, 20 mM DTT, 1 mg/ml BSA, 10 mMspermidine.

10× Kinasing Buffer: 0.5M Tris pH 7.5, 10 mM ATP, 20 mM DTT, 10 mMspermidine, 1 mg/ml BSA 100 mM MgCl₂.

By "vector" is meant a DNA molecule, derived from a plasmid orbacteriophage, into which fragments of DNA may be inserted or cloned. Avector will contain one or more unique restriction sites, and may becapable of autonomous replication in a defined host or vehicle organismsuch that the cloned sequence is reproducible. Thus, by "DNA expressionvector" is meant any autonomous element capable of replicating in a hostindependently of the host's chromosome, after additional sequences ofDNA have been incorporated into the autonomous element's genome. SuchDNA expression vectors include bacterial plasmids and phages.

Preferred for the purposes of the present invention, however, are viralvectors, such as those derived from simian virus strain 40 (SV40). SV40is a papovavirus having a molecular weight of 28Mdal, and containing acircular double-stranded DNA molecule having a molecular weight of3Mdal, which comprises the entire genome of the virus. The entirenucleotide sequence of this single, small, covalently closed circularDNA molecule has been determined. Fiers et al., Nature 273:113-120(1978); Reddy et al., Science 200:494-502 (1978). The viral DNA of SV40may be obtained in large quantities, and the genomic regions responsiblefor various viral functions have been accurately located with respect toa detailed physical map of the DNA. Fiers et al., supra; Reddy et al.,supra. The viral genome of SV40 can multiply vegetatively or as anintegral part of cellular chromosomes, and a wealth of informationexists on the replication and expression of this genome.

Also preferred for the purposes of the present invention is asingle-stranded bacteriophage cloning vehicle, designated M13, having aclosed circular DNA genome of approximately 6.5 kb. An advantage ofutilizing M13 as a cloning vehicle is that the phage particles releasedfrom infected cells contain single-stranded DNA homologous to only oneof the two complementary strands of the cloned DNA, which therefore canbe used as a template for DNA sequencing analysis.

Even more preferred for the purposes of the present invention are theexpression vectors designated piH3, piH3M, and CDM8. CDM8 was depositedat the ATCC on Feb. 24, 1988, and has accession number ATCC 67635.

By "tissue culture" is meant the maintenance or growth of animal tissuecells in vitro so as to allow further differentiation and preservationof cell architecture or function or both. "Primary tissue cells" arethose taken directly from a population consisting of cells of the samekind performing the same function in an organism. Treating such tissuecells with the proteolytic enzyme trypsin, for example, dissociates theminto individual primary tissue cells that grow well when seeded ontoculture plates at high densities. Cell cultures arising frommultiplication of primary cells in tissue culture are called "secondarycell cultures." Most secondary cells divide a finite number of times andthen die. A few secondary cells, however, may pass through this "crisisperiod", after which they are able to multiply indefinitely to form acontinuous "cell line." Cell lines often will contain extra chromosomes,and usually are abnormal in other respects as well. The immortality ofthese cells is a feature shared in common with cancer cells.

Preferred cell lines for use as tissue culture cells according to thepresent invention include the monkey kidney cell line, designated "COS."COS cells are those that have been transformed by SV40 DNA containing afunctional early gene region but a defective origin of viral DNAreplication. COS cell clone M6 is particularly preferred for useaccording to the method of the invention. Also preferred for thepurposes of the present invention are murine "WOP" cells, which are NIH3T3 cells transfected with polyoma origin deletion DNA. cDNA may beintroduced into the host tissue culture cells of the present inventionby any methods known to those of skill. Transfection may be accomplishedby, for example, protoplast fusion, by spheroplast fusion, or by theDEAE dextran method (Sussman et al., Cell. Biol. 4:1641-1643 (1984)).

If spheroplast fusion is employed, a preferred method is the followingvariant based on Sandri-Goldrin et al., Mol. Cell Bio. 1:743-752 (1981).Briefly, for example, a set of six fusions requires 100 ml of cells inbroth. Grow cells containing amplifiable plasmid to OD 600=0.5 in LB.Add spectinomycin to 100 μg/ml (or chloramphenicol to 150 μg/ml).Continue incubation at 37° C. with shaking for 10-16 hours. (Cells beginto lyse with prolonged incubation in spectinomycin or chloramphenicolmedium). Spin down 100 ml of culture (JA14/GSA rotor, 250 ml bottle) 5min. at 10,000 rpm. Drain well, resuspend pellet in bottle with 5 mlcold 20% sucrose, 50 mM Tris-HCL pH 8.0. Incubate on ice 5 min. Add 2 mlcold 0.25M EDTA pH 8.0, incubate 5 min. at 37° C. (waterbath). Place onice, check percent conversion to spheroplasts by microscopy. In flowhood, slowly add 20 ml of cold DME/10% sucrose/10 mM MgCl₂ (dropwise,ca. 2 drops per second). Remove media from cells plated the day beforein 6 cm dishes (50% confluent). Add 5 ml of spheroplast suspension toeach dish. Place dishes on top of tube carriers in swinging bucketcentrifuge. Up to 6 dishes can be comfortably prepared at once. Dishescan be stacked on top of each other, but 3 in a stack is not advisableas the spheroplast layer on the top dish is often torn or detached aftercentrifugation. Spin at 1000×g 10 min. Force is calculated on the basisof the radius to the bottom plate. Aspirate fluid from dishes carefully.Pipet 1.5-2 ml 50% (w/w) PEG 1450 (or PEG 1000)/500% DME (no serum) intothe center of the dish. If necessary, sweep the pipet tip around toensure that the PEG spreads evenly and radially across the whole dish.After PEG has been added to the last dish, prop all of the dishes up ontheir lids so that the PEG solution collects at the bottom. Aspirate thePEG. The thin layer of PEG that remains on the cells is sufficient topromote fusion; the layer remaining is easier to wash off, and bettercell viability can be obtained, than if the bulk of the PEG is leftbehind. After 90 to 120 seconds (PEG 1000) or 120 to 150 seconds (PEG1450) of contact with the PEG solution, pipet 1.5 ml of DME (no serum)into the center of the dish. The PEG layer will be swept radially by theDME. Tilt the dishes and aspirate. Repeat the DME wash. Add 3 ml ofDME/10% serum containing 15 μg/ml gentamicin sulfate. Incubate 4-6 hoursin incubator. Remove media and remaining bacterial suspension, add moremedia and incubate 2-3 days. Extensive washing of the cell layer toremove PEG tends to remove many of the cells without any substantialbenefit. If the cells are allowed to sit in the second DME wash for afew minutes, most of the spheroplast layer will come up spontaneously;however it is preferred to wash briefly and allow the layer to come offin the complete medium at 37° C.

The PEG solution can be conveniently prepared by melting a fresh bottleof PEG at 60° C. and pouring approximate 50 ml aliquots by means of a 50ml centrifuge tube into preweighed bottles. The aliquoted PEG is storedat 5° C. in the dark. To make up a fresh bottle, weigh the aliquot,remelt, and add an equal volume of DME (no serum). Adjust the pH with7.5% Na bicarbonate solution if necessary, and filter sterilize. Theresulting PEG solution may be stored up to 3 months at room temperaturewithout detectable adverse consequence.

Transfected host cells will be cultured according to the invention inorder to accomplish expression of the protein encoded by the cDNA clone,and to increase the absolute numbers of cells available for subsequentimmunoselection. Those skilled in the art will know of appropriatemethods and media for this purpose, taking into account the cell typeand other variables routinely considered. COS cells, for example, may becultured in Dulbecco's modified Eagle's medium (DME) supplemented with10% calf serum and gentamycin sulfate. Transient expression oftransfected cells normally can be expected between 48 and 72 hoursposttransfection. However, this time period may vary depending upon thetype or strain of host cell used and the cell culture conditions, aswill be apparent to those of ordinary skill.

Immunoprecipitation, blotting, and cDNA sequencing of genes clonedaccording to the methods of the present invention may be carried out byany convenient methods known to those of skill. For example, theimmunoprecipitation protocol of Clark et al., Leukocyte Typing II, Vol.II, pp. 155-167 (1986), is preferred. Southern, Northern, or other blotanalysis methods known to those of skill may be employed, usinghybridization probes prepared by known methods, such as that of Hu etal. (Gene 18:271-277 (1982)). cDNA sequencing also may be accomplishedby known methods, including the dideoxynucleotide method of Sanger etal., Proc. Natl. Acad. Sci. (USA) 74:5463-5467 (1977).

The antibodies used according to the present invention may be polyclonalor monoclonal. These may be used singly, or in conjunction with otherpolyclonal or monoclonal antibodies to effect immunoselection of cellsexpressing the desired antigen or antigens by the methods of the presentinvention. Methods of preparing antibodies or fragments thereof for useaccording to the present invention are known to those of skill.

Standard reference works setting forth general principles of immunologyinclude Klein, J., Immunology: The Science of Self-NonselfDiscrimination, John Wiley & Sons, publisher, New York (1982); Kennett,R., et al., eds., Monoclonal Antibodies, Hybridoma: A New Dimension inBiological Analyses, Plenum Press, publisher, New York (1980); Campbell,A., "Monoclonal Antibody Technology" in Burden, R., et al., eds.,Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13,Elsevere, publisher, Amsterdam (1984).

The term "antibody" is meant to include the intact molecule as well asfragments thereof, such as, for example, Fab and F(ab)'₂ fragments,which also are able to bind to antigen. Polyclonal antibody preparationsmay be derived directly from the blood of the desired animal speciesafter immunization with the antigen of interest, or a fragment thereof,using any of the standard protocols known to those of ordinary skill.Similarly, monoclonal antibodies may be prepared using known methods(Kohler et al., Eur. J. Immunol. 6:292 (1976)). Use of monoclonalantibodies is preferred for the purposes of the present invention.

For the purposes of immunoselection according to the present invention,the tissue culture host cells which have been exposed to antibodiesdirected against the target cell surface antigen are separated from hostcells which do not express the target antigen by distributing the cellsonto a substrate coated with antibody directed against the antibody forthe antigen. This technique, termed "panning," will be known to those ofskill, and is described, for example, by Mage et al., J. Immunol. Meth.15:47-56 (1977), and Wysocki and Sato, Proc. Natl. Acad. Sci. (USA)75:2844-2848 (1978).

Panning according to the methods of the present invention may be carriedout as follows:

a. Antibody-coated dishes. Bacteriological 60 mm plates, Falcon 1007 orequivalent, or 10 cm dishes such as Fisher 8-757-12 may be used. Sheepanti-mouse affinity purified antibody (from, for example, CooperBioMedical (Cappell)) is diluted to 10 μg/ml in 50 mM Tris HCl, pH 9.5.Add 3 ml per 6 cm dish, or 10 ml per 10 cm dish. Let sit ca. 1.5 hrs.,remove to next dish 1.5 hrs., then to 3rd dish. Wash plates 3× with0.15M NaCl (a wash bottle is convenient for this), incubate with 3 ml 1mg/ml BSA in PBS overnight, aspirate and freeze.

b. Panning. Cells will be in 60 mm dishes. Aspirate medium from dish,add 2 ml PBS/0.5 mM EDTA0.02% azide and incubate dishes at 37° C. for 30min. to detach cells from dish. Triturate cells vigorously with shortpasteur pipet, and collect cells from each dish in a centrifuge tube.Spin 4 min. setting 2.5 (200×g) (takes 5 min). Resuspend cells in0.5-1.0 ml PBS/EDTA/azide/5% FBS and add antibodies. Incubate at least30 min. on ice. Add an equal volume of PBS/EDTA/azide, layer carefullyon 3 ml PBS/EDTA/azide/2% Ficoll, and spin 4 min. at setting 2.5.Aspirate supernatant in one smooth movement. Take up cells in 0.5 mlPBS/EDTA/azide and add aliquots to antibody-coated dishes containing 3ml PBS/EDTA/azide/5% FBS by pipetting through 100 micron Nylon mesh(Tetko). Add cells from at most two 60 mm dishes to one 60 mmantibody-coated plate. Let sit at room temperature 1-3 hours. Removeexcess cells not adhering to dish by gentle washing with PBS/5% serum orwith medium. 2 or 3 washes of 3 ml are usually sufficient.

c. Hirt Supernatant. A preferred variant of the method of Hirt, J.Molec. Biol. 26:365-369 (1967), is as follows: Add 0.4 ml 0.6% SDS, 10mM EDTA to panned plate. Let sit 20 minutes (can be as little as 1 min.if there are practically no cells on the plate). Pipet viscous mixtureinto microfuge tube. Add 0.1 ml 5M NaCl, mix, put on ice at least 5 hrs.Keeping the mixture as cold as possible seems to improve the quality ofthe Hirt. Spin 4 min., remove supernatant carefully, phenol extract(twice if the first interface is not clean), add 10 ug linearpolyacrylamide (or other carrier), fill tube to top with EtOH,precipitate, and resuspend in 0.1 ml. Add 3 volumes EtOH/NaOAc,reprecipitate and resuspend in 0.1 ml. Transform into MC1061/p3,preferably using the high efficiency protocol hereinafter described. Ifthe DNA volume exceeds 2% of the competent cell aliquot, thetransformation efficiency will suffer. 5% gives the same number ofcolonies as 2.5% (efficiency is halved).

It is preferred for this aspect of the present invention to use"blockers" in the incubation medium. Blockers assure that non-specificproteins, proteases, or antibodies present do not cross-link with ordestroy the antibodies present on the substrate or on the host cellsurface, to yield false positive or false negative results. Selection ofblockers can substantially improve the specificity of theimmunoselection step of the present invention. A number of non-specificmonoclonal antibodies, for example, of the same class or subclass(isotype) as those used in the immunoselection step (e.g., IgG₁, IgG₂ A,IgGm, etc.) can be used as blockers. Blocker concentration (normally1-100 μg/μl) is important to maintain the proper sensitivity yet inhibitunwanted interference. Those of skill also will recognize that thebuffer system used for incubation may be selected to optimize blockingaction and decrease non-specific binding.

A population of cells to be panned for those expressing the target cellsurface antigen is first detached from its cell culture dish (harvested)without trypsin. The cells then are exposed to a first antibody, whichmay be polyclonal or monoclonal, directed against the antigen ofinterest or against a family of related antigens. At this initial stage,a single antibody or a group of antibodies may be used, the choicedepending upon the nature of the target antigen, its anticipatedfrequency, and other variables that will be apparent to those of skill.Target antigens expressed on the surfaces of host cells will form anantigen-antibody complex.

The cells subsequently are placed in close apposition to a substrate,such as a culture dish, filter disc, or the like, which previously hasbeen coated with a second antibody or group of antibodies. This secondantibody will be directed against the first antibody, and its choicewill be a matter of ordinary skill dictated by, for example, the animalin which the first antibody was raised. For example, if the firstantibody was raised in mice, the second antibody might be directedagainst mouse immunoglobulins, raised in goats or sheep. Cellsexpressing the target antigen will adhere to the substrate via thecomplex formed between the antigen, the first antibody, and the secondantibody. Adherent cells then may be separated from nonadherent cells bywashing. DNA encoding the target antigen is prepared from adherent cellsby known methods, such as that of Hirt, J. Molec. Biol. 26:365-369(1967). This DNA may be transformed into E. coli or other suitable hostcells for further rounds of fusion and selection, to achieve the desireddegree of enrichment.

In the usual case, the initial rounds of immunoselection will employ apanel of first antibodies directed against an epitope or group ofepitopes common to the family of antigens to which the target antigenbelongs. This will be sufficient to narrow the number of clones forfuture rounds quite significantly. Two such rounds usually will be foundadequate, but the number of rounds may vary as mentioned above.Thereafter, a single round of selection may be performed employing asingle first antibody or a group of first antibodies recognizing onlythe target antigen.

By "substrate" is meant a solid surface to which antibodies may be boundfor immunoselection according to the present invention. Known suitablesubstrates include glass, polystyrene, polypropylene, dextran, nylon,and other materials. Tubes, beads, microtiter plates, bacteriologicalculture dishes, and the like formed from or coated with such materialsmay be used. Antibodies may be covalently or physically bound to thesubstrate by known techniques, such as covalent bonding via an amide orester linkage, or by absorption. Those skilled in the art will know manyother suitable substrates and methods for immobilizing antibodiesthereupon, or will be able to ascertain such substrates and methodsusing no more than routine experimentation.

The choice of host tissue culture cells for use according to the presentinvention preferably should be such as to avoid the situation in whichthe antibodies used for panning recognize determinants on untransfectedcells. Thus, while COS cells are preferred for transient expression ofcertain surface antigens, more preferred are murine WOP cells. Of thelatter, WOP 3027 cells are even more preferred. WOP cells allowvirtually all antibodies to be used, since cross-reactions betweenmurine antibodies and murine cell surface determinants are rare.

The insert size of the recombinant DNA molecule should be chosen tomaximize the likelihood of obtaining an entire coding sequence. Those ofskill will know various methods by which a preliminary determination ofoptimal insert size for a given gene may be determined.

Vector construction and cDNA insertion

Vectors suitable for expression of cDNA in mammalian tissue culturecells may be constructed by known methods. Preferred for the purposes ofthe present invention is an expression vector containing the SV40origin. The vector may contain a naturally derived or synthetictranscription origin, and the SV40 early region promoter. Even morepreferred is a chimeric promoter composed of human cytomegalovirusimmediate early enhancer sequences. Various "enhancer sequences" alsomay be used with SV40 vectors. These are described, for example, byBanerji et al., Cell 27:299-308 (1981); Levinson et al., Nature295:568-572 (1982); and Conrad et al., Mol. Cell. Biol. 2:949-965(1982).

Insertion of cDNA into the vectors of the present invention can occur,for example, by homopolymeric tailing with terminal transferase.However, homopolymeric tracts located 5' to cDNA inserts may inhibit invitro and in vivo expression. Thus, preferred for purposes of thepresent invention is the use of inverted identical cleavage sitesseparated by a short replaceable DNA segment. Such inverted identicalcleavage sites, preferably employing the BstXI restriction endonuclease,may be used in parallel with cDNA synthetic oligonucleotides, y givingthe same termini as the replaceable segment of the vector. In thismanner, the cDNA cannot ligate to itself, but can ligate to the vector.This allows the most efficient use of both cDNA and vector.

Another embodiment of the present invention is the above-describedefficient oligonucleotide-based strategy to promote cDNA insertion intothe vector. The piH3M vector of the present invention is preferred, andemploys the inverted endonuclease sites. This vector may contain an SV40origin of replication, but a more preferred form contains an M13 origin.This vector, containing the M13 origin, allows high level expression inCOS cells of coding sequences placed under its control. Also, the smallsize and particular arrangement of sequences in the plasmid permit highlevel replication in COS cells.

By "cell surface antigen" is meant any protein that is transportedthrough the intracellular membrane system to the cell surface. Suchantigens normally are anchored to the cell surface membrane through acarboxyl terminal domain containing hydrophobic amino acids that lie inthe lipid bilayer of the membrane, and there exert their biological andantigenic effects. Antigens such as those of T-lymphocytes areparticularly suited for gene cloning by the method of the presentinvention. However, cell surface antigens of any cells may be clonedaccording to the present method. Moreover, proteins not normallyexpressed on the cell surface may admit of cloning according to thepresent method by, for example, using fluorescence activated cellsorting (FACS) to enrich for fixed cells expressing intracellularantigens.

By "substantially pure" is meant any antigen of the present invention,or any gene encoding any such antigen, which is essentially free ofother antigens or genes, respectively, or of other contaminants withwhich it might normally be found in nature, and as such exists in a formnot found in nature.

By "functional derivative" is meant the "fragments," "variants,""analogs," or "chemical derivatives" of a molecule. A "fragment" of amolecule, such as any of the antigens of the present invention, is meantto refer to any polypeptide subset of the molecule containing afunctional domain such as an epitope, a ligand binding site, anextracellular domain or an immunoglobulin domain, which comprises atleast about 6 amino acids. A "variant" of such molecules is meant torefer to a naturally occurring molecule substantially similar to eitherthe entire molecule, or a fragment thereof. An "analog" of a molecule ismeant to refer to a non-natural molecule substantially similar to eitherthe entire molecule or a fragment thereof. As used herein, a molecule issaid to be a "chemical derivative" of another molecule when it containsadditional chemical moieties not normally a part of the molecule. Suchmoieties may improve the molecule's solubility, absorption, biologicalhalf life, etc. The moieties may alternatively decrease the toxicity ofthe molecule, eliminate or attenuate any undesirable side effect of themolecule, etc. Moieties capable of mediating such effects are disclosed,for example, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

A fragment, variant, analog and/or chemical derivative of a subjectantigen is said to be a "functional derivative" of the antigen if theamino acid sequence of the former has at least about 80% identity to thesequence of the latter, and if the former has at least about 30% of abiological activity or function of the latter. Increasingly preferredare amino acid identities that increase integrally, i.e., at least about81%, 82%, etc. identity. Also, increasingly preferred biologicalactivities are those of at least about 40%, 50%, 60%, 70%, 80%, and 90%.

A nucleotide sequence is said to be a "functional derivative" of adisclosed nucleotide sequence encoding an antigen if the former encodesa disclosed antigen or a functional derivative thereof.

Biological activities are those operations, functions or processes whichare characteristic of living organisms. Biological activities can alsoinclude the reproduction, extension or adaptation of living processes toin vitro or non-natural systems, such as the biological activityexhibited when an antigen or its functional derivative is artificiallyintroduced into a test animal to induce the production of antibodies. Anantigen can have one or more biological activities. Biologicalactivities can be detected or measured by methods or assays that arecharacteristic for that activity. For a functional derivative to have abiological activity substantially the same as that of an antigen, itmust have a biological activity of at least about 30% of that of antigenas measured by an assay characteristic for that activity and known tothose of skill in the art.

The substantially pure antigens that have been expressed by methods ofthe present invention may be used in immunodiagnostic assay methods wellknown to those of skill, including radio-immunoassays (RIAs), enzymeimmunoassays (EIAs) and enzyme-linked immunosorbent assays (ELISAs). Thesubstantially pure proteins of the present invention, in soluble form,may be administered alone or in combination with other antigens of thepresent invention, or with other agents, including lymphokines andmonokines or drugs, for the treatment of immune-related diseases anddisorders in animals, including humans. As examples of such disordersthat may benefit from treatment with the substantially pure proteins ofthe present invention may be mentioned immune deficiency diseases,diseases of immediate type hypersensitivity, asthma, hypersensitivitypneumonitis, immune-complex disease, vasculitis, systemic lupuserythematosus, rheumatoid arthritis, immunopathogenic renal injury,acute and chronic inflammation, hemolytic anemias, platelet disorders,plasma and other cell neoplasms, amyloidosis, parasitic diseases,multiple sclerosis, Guillain-Barre syndrome, acute and subacutemyopathic paralysis, myasthenia gravis, immune endocrinopathies, andtissue and organ transplant rejection, all as described in Petersdorf etal., eds., Harrison's Principles of Internal Medicine, supra. See alsoWeir, ed., supra; Boguslaski et al., eds., supra; and Holborow et al.,eds., supra.

When used for immunotherapy, the antigens of the present invention maybe unlabeled or labeled with a therapeutic agent. Examples oftherapeutic agents which can be coupled to the antigens of the inventionfor immunotherapy are drugs, radioisotopes, lectins, and toxins.

The dose ranges for the administration of the antigens of the presentinvention are those large enough to produce the desiredimmunotherapeutic effect, but not so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage employed will vary with the age,condition, sex, and extent of the disease in the patient.Counterindications (if any), immune tolerance and other variables alsowill affect the proper dosage. Administration may be parenteral, byinjection or by gradual perfusion over time. Administration also may beintravenous, intraparenteral, intramuscular, subcutaneous, orintradermal.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Examples ofnon-aqueous solvents include propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic and aqueoussolutions, emulsions, or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives also may be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like. Such preparations, and the manner and method ofmaking them, are known and described, for example, in Remington'sPharmaceutical Science, 16th ed., supra.

The antigens of the present invention also may be prepared asmedicaments or pharmaceutical compositions comprising the antigens,either alone or in combination with other antigens or other agents suchas lymphokines, monokines, and drugs, the medicaments being used fortherapy of animal, including human, immune-related indications.

Although the antigens of the present invention may be administeredalone, it is preferred that they be administered as a pharmaceuticalcomposition. The compositions of the present invention comprise at leastone antigen or its pharmaceutically acceptable salt, together with oneor more acceptable carriers and optionally other therapeutic agents. By"acceptable" is meant that the agent or carrier be compatible with otheringredients of the composition and not injurious to the patient.Compositions include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal, or parenteraladministration. The compositions conveniently may be presented in unitdosage form, and may be prepared by methods well known in thepharmaceutical arts. Such methods include bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general, compositions are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers, or both, andshaping the product formed thereby, if required.

Orally administered pharmaceutical compositions according to the presentinvention may be in any convenient form, including capsules, cachets, ortablets, each containing a predetermined amount of the activeingredient. Powders or granules also are possible, as well as solutionor suspension in aqueous or nonaqueous liquids, or oil-in-water liquidemulsions, or water-in-oil liquid emulsions. The active ingredient alsomay be presented as a bolus, electuary or paste.

Having now described the invention, the same will be more fullyunderstood by reference to the following examples, which are notintended in any way to limit the scope of the invention.

EXAMPLE I Isolation, Molecular Cloning, and Structure of the Human CD2Antigen

The cDNA expression vector piH3

A COS cell expression vector was constructed from piSV (Little et al.,Mol. Biol. Med. 1:473-488 (1983)) by inserting a synthetic transcriptionunit between the suppressor tRNA gene and the SV40 origin. Thetranscription unit consisted of a chimeric promoter composed of humancytomegalovirus AD169 immediately early enhancer sequences fused to theHIV LTR -67 to +80 sequences. Immediately downstream from the LTR +80sequence was inserted a polylinker containing two BstXI sites separatedby a 350 bp stuffer; the BstXI sites were flanked by Xbal sites, whichcould also be used to excise the insert. Downstream from the polylinkerwere placed the SV40 small t antigen splice and early regionpolyadenylation signals derived from pSV2. The nucleotide sequence ofthe vector is shown in FIGS. 1A-1B.

cDNA library construction

RNA was prepared from HPB-ALL cells by the guanidinium thiocyanate/CsClmethod, as described above. PolyA⁺ RNA was prepared from total RNA byoligo dT selection. Maniatis et al. Molecular Cloning: A LaboratoryManual, supra. cDNA was synthesized by the method of Gubler and Hoffman(Gene 25:263-269 (1982)). BstXI adaptors were ligated to the cDNA, andthe reaction products fractionated by centrifugation through a 5 ml-20%potassium acetate gradient containing 1 mM EDTA for 3 hours at 50 k rpmin a SW55 rotor. 0.5 ml fractions were collected manually through asyringe needle or butterfly inserted just above the curve of the tube.Individual fractions were ethanol-precipitated after addition of linearpolyacrylamide (Strauss and Varshavsky, Cell 37:889-901 (1984)) to 20μg/ml. Fractions containing cDNA larger than 700 bp were pooled andligated to gradient purified BstXI digested piH3 vector.

The ligated DNA was transformed into E. coli MC1061/p3 made competent bythe following protocol: The desired strain was streaked out on an LBplate. The next day a single colony was inoculated into 20 ml TYM broth(recipes below) in a 250 ml flask. The cells were grown to midlog phase(OD₆₀₀ about 0.2-0.8), poured into a 2 l flask containing 100 ml TYM,and vigorously agitated until cells grew to 0.5-0.9 OD, then dilutedagain to 500 ml in the same vessel. When the cells grew to OD₆₀₀ 0.6,the flask was placed in ice-water, and shaken gently to assure rapidcooling. When the culture was cool, it was spun at 4.2 k rpm for 15minutes (J6). The supernatant was poured off and the pellet resuspendedin about 100 ml cold TfB I (below) by gentle shaking on ice. Thereafter,it was respun in the same bottle at 4.2 k rpm for 8 minutes (J6). Thesupernatant was poured off and the pellet resuspended in 20 ml cold TfBII by gentle shaking on ice. 0.1 to 0.5 ml aliquots were placed inprechilled microfuge tubes, frozen in liquid nitrogen, and stored at-70° C. For transformation, an aliquot was removed, thawed at roomtemperature until just melting, and placed on ice. DNA was added, letsit on ice 15-30 minutes, and incubated at 37° C. for 5 minutes (6minutes for 0.5 ml aliquots). Thereafter the DNA-containing suspensionswere diluted 1:10 in LB and grown for 90 minutes before plating orapplying antibiotic selection. Alternatively, the heat-pulsedtransformation mix was plated directly on antibiotic plates onto which athin (4-5 ml) layer of antibiotic-free LB agar was poured just beforeplating.

Media and Buffers: TYM: 2% Bacto-Tryptone, 0.5% Yeast Extract, 0.1MNaCl, 10 mM MgSO₄ (can be added before autoclaving). TfB I: 30 mM KOAc,50 mM MnCl₂, 100 mM KCL, 10 mM CaCl₂, 15% (v/v) glycerol. TfB II: 10 mMNa-MOPS, pH 7.0, 75 MM CaCl₂, 10 mM KCl, 15% glycerol.

Recovery of cDNA clones by panning

Bacteriological culture dishes (Falcon 1007) were prepared for panningby coating with an affinity purified sheep anti-mouse IgG antibody asdescribed by Wysocki and Sato (Proc. Natl. Acad. Sci. USA 75:2844-2848(1978)), except that dishes were washed with 0.15M NaCl from a washbottle instead of PBS, and unreacted sites were blocked by overnightincubation in PBS containing 1 mg/ml BSA. Dishes were typically preparedin large batches and stored frozen, after aspiration of the PBS/BSA. Inthe first round of screening, 24 6 cm dishes of 50% confluent COS cellswere transfected by protoplast fusion according to the method ofSandri-Goldrin et al., Mol. Cell Biol. 1:743-752 (1981). 72 hours postfusion the cells were detached by incubation in PBS/1 mM EDTA/0.02%sodium azide at 37° C. for 30 minutes. The detached cells were pooled,centrifuged, and resuspended in cold PBS/EDTA/5% Fetal Bovine Serumcontaining monoclonal antibodies, usually as ascites at 1:1000 dilution,but also as commercial reagents at the concentrations suggested by themanufactures. After 1 hour on ice, the cells were diluted with 1:1 withPBS/EDTA/azide and layered on 10 ml of PBS/EDTA/azide containing 2%Ficoll 400. After centrifugation (400×g, 5 minutes), the supernatant wascarefully aspirated, the pellet resuspended in a small amount ofPBS/EDTA/5% FBS, and the cells distributed into panning platescontaining 3 ml of PBS/EDTA/5% FBS. The plates were then treatedessentially as described by Wysocki and Sato, Proc. Natl. Acad. Sci. USA75:2844-2848 (1978). Episomal DNA was recovered from the adherent cellsby the Hirt (J. Mol. Biol. 26:365-269 (1967)) procedure and transformedinto MC1061/p3.

Cell lines and cell culture

COS cell clone M6 cells were propagated in Dulbecco's modified Eagle'smedium supplemented with 10% calf serum and gentamycin sulfate at 15μg/ml (DME/10% calf serum). Cells were split the day before transfectionin 6 cm dishes at approximately 1:8 ratio from stock plates kept asdense as possible without overtly affronting the cells. T cell lineswere grown in Iscove's modification of Dulbecco's medium (IMDM)containing gentamycin as above, and either NuSerum (CollaborativeResearch) or fetal bovine serum at 10%.

COS cell transfection for immunofluorescence studies

COS cells at 50% confluence in 6 cm dishes were transfected in a volumeof 1.5 ml with a cocktail consisting of DME or IMDM medium containing10% NuSerum (Collaborative Research), 400 μg/ml DEAE Dextran, 10 μMchloroquine diphosphate, and 1 μg/ml DNA. After 4 hours at 37° C. (orearlier if the cells appeared ill), the transfection mix was removed andthe cells were treated with 10% DMSO in PBS for 2 minutes. Sussman andMilman, Cell Biol. 4:1641-1643 (1984). Cells were then returned toDME/10% calf serum for 48 to 72 hours to allow expression.

Immunoprecipitations, Northerns and Southerns

T cells were labeled by lactoperoxidase treatment, lysed, andimmunoprecipitated by the procedure of Clark and Einfeld (LeukocyteTyping II, Vol. II, pp. 155-167 (1986)), using commercially availablegoat anti-mouse IgG agarose beads (Cooper Biomedical). COS cells weretransfected by DEAE Dextran method and trypsinized and passed withoutdilution into new plates 24 hours after transfection. 36 hours later,cells were detached by exposure to PBS/EDTA as above, centrifuged, andlabeled by the lactoperoxidate method. A cleared lysate was prepared asfor the T cell immunoprecipitations, except that the lysis buffercontained 1 mM PMSF, and incubation with the primary antibody wascarried out for only 2 hours at 4° C. Eluted samples were fractionatedon discontinuous 11.25% polyacrylamide gels using the buffer system ofLaemmli (Nature 227:680-685 (1970)).

Northern blot analysis was carried out essentially as described(Maniatis et al., Molecular Cloning, a Laboratory Manual (1982)), exceptthat DMSO was omitted from the loading buffer, denaturation was at 70°C. for 5 minutes, and the gel contained 0.6% formaldehyde rather than6%. The gel was stained in two volumes of water containing 1 μg/mlethidium bromide, photographed, and transferred to nylon (GeneScreen,DuPont) in the staining liquor. The transferred RNA was irradiated byexposure to a germicidal lamp through Saran Wrap (Church and Gilbert,Proc. Natl. Acad. Sci. USA 8:1991-1995 (1984)) for 5 minutes at a flux(measured at 254 nm) of 0.22 mW/cm². Southern blot analysis was carriedout by alkaline transfer to nylon (GeneScreen, DuPont) as described byReed and Mann (Nucl. Acids Res. 13:7207-7221 (1986)). Hybridizationprobes were prepared by the method of Hu and Messing (Gene 18:271-277(1982)), and blots were prehybridized in SDS/phosphate buffer (Churchand Gilbert, Proc. Natl. Acad. Sci. USA 8:1991-1995 (1984)) containing10 DNA microgram equivalents of M13 mp19 phage.

Erythrocyte Rosetting

Erythrocytes were prepared from whole blood by three centrifugations inPBS. COS cells were transfected in 6 cm dishes with CD2 or other surfaceantigen expression clones by the DEAE method. 48 to 72 hoursposttransfection, the medium was aspirated and 2 ml of PBS/5% FDS/azidewas added to each plate, followed by 0.4 ml of the appropriateerythrocyte samples as 20% suspensions in PBS. After 1 hour at roomtemperature, the nonadherent erythrocytes were gently washed off, andthe plates examined.

A cDNA encoding CD2 antigen determinants was isolated in the followingmanner: cDNA was prepared from RNA extracted from the human T Cell tumorline HPB-ALL and inserted into the SV40 origin-based expression vectorpiH3 as described above. A cDNA library of approximately 3×10⁵recombinants was constructed, and the library was introduced into COScells by protoplast fusion. Three days later the cells were detached byexposure to EDTA and treated with a pool of monoclonal antibodies,including three (OKT11, Leu5b, and Coulter T11) directed against CD2determinants. The antibody-treated cells were distributed into dishescoated with an affinity purified sheep anti-mouse IgG antibody, allowedto attach, and separated from the nonadherent cells by gentle washing.This method of enrichment is known in the immunological literature (Mageet al. J. Immunol. Methods 15:47-56 (1977).

The resulting colonies were pooled, fused into COS cells, and subjectedto a second round of panning as before. In the third round, a portion ofthe detached cells was treated with a mixture of three monoclonalantibodies specific for CD2, and a Hirt supernatant was again generatedand transformed into E. coli. DNA was prepared from eight of theresulting colonies and transfected into COS cells. After three days,surface expression of the CD2 antigen was detected by indirectimmunofluorescence in six of eight transfected dishes. Restrictionenzyme digestion of the corresponding plasmid DNAs revealed a 1.5 kbinsert in all six isolates.

One of the six clones was prepared in larger quantities for furtheranalysis. Following transfection into COS cells, indirectimmunofluorescence analysis with a partial panel of antibodies providedby the Third International Workshop on Leukocyte DifferentiationAntigens showed that all of the antibodies provided gave positivereactions with the exception of one sample which also failed to reactwith phytohemagglutinin-activated T lymphocytes. Among the 17 antibodiestested were at least eight distinguishable groups defined by theirdiffering patterns of reactivity with lymphocytes of various primatespecies. Jonker and Nooij, Leukocyte Typing II, Vol. I, pp. 373-387(1986).

cDNA sequence analysis

The CD2 cDNA insert was subcloned into M13 mp19 (Vieira and Messing,Gene 19:259-268 (1982)) in both orientations, and the sequencedetermined by the dideoxynucleotide method (FIGS. 2A and 2B). Sanger etal., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977). An open readingframe was observed to extend 360 residues from an ATG triplet satisfyingthe consensus criteria of Kozak (Microbiol. Rev.: 1-47:45 (1983)) fortranslational initiation codons (FIG. 1). The predicted amino acidsequence evokes an integral membrane protein with a single membranespanning hydrophobic anchor terminating in a rather largeintracytoplasmic domain. Comparison of the N-terminal amino sequencewith the matrix of signal sequence residue frequencies constructed byvon Heijne (Nucl. Acids Res. 14:4683-4690 (1986)) suggests that matureCD2 peptide is formed by cleavage of a precursor peptide between the19th (Ser) and 20th (Lys) residues.

A surprising and unexpected feature of this sequence is the presence ofa potential N-linked glycosylation site just proximal to the proposedcleavage site. The resulting polypeptide backbone has a predictedmolecular weight of 38.9 kd divided into an external domain of mass 21.9kb and a cytoplasmic domain of mass 14.6 kd. Three N-linkedglycosylation sites are present in the extracellular domain.

The membrane spanning domain comprises 26 unchanged residues ofpredominantly hydrophobic character. In the nine residues immediatelyfollowing are seven basic residues, either lysines or arginines. Theappearance of predominantly hydrophobic residues followed by basicresidues is a common organizational feature of transmembrane proteinsbearing carboxyl-terminal anchors.

Another surprising feature of the transmembrane domain is the appearanceof a cys-gly-gly-gly, a beta turn motif (Chou and Fasman, Annual Reviewof Biochemistry 47:251-276 (1978)), flanked by hydrophobic residues(which are frequently found flanking beta turns). Because only 20residues arrayed in an alpha helix are theoretically needed to traversethe 3 nm membrane bilayer (Tanford, Science 200:1012-1018 (1978)), andas few as 14 hydrophobic residues can allow insertion and export of anintegral membrane protein (Adams and Rose, Cell 41:1007-1015 (1985)),the transmembrane segment of the CD2 antigen may contain a bend or kink.

The rather large size of the cytoplasmic domain leaves open thepossibility that CD2 possesses an intrinsic enzymatic activity. Thecytoplasmic domain is very rich in prolines and contains three siteswith high turn probability.

Comparison of the amino acid sequence with the NBRF database revealed nosubstantive homologies with other proteins. In particular, no homologywith the T cell receptor alpha or beta chains was observed, ruling outthe suggestion that CD2 is a primordial T cell receptor. Milanese etal., Science 231:1118-1122 (1986).

Just inside the cytoplasmic face of the protein is a run of basicproteins followed by a serine residue, a pattern found at the samelocation in both the EGF receptor and the class I histocompatibilitygenes, and in each case a known site for either in vivo (EGF) and invitro (HLA) phosphorylation by protein kinase C or cyclic AMP-dependentprotein kinase, respectively. Hunter et al., Nature 311:480-482 (1984);Davis and Czech, Proc. Natl. Acad. Sci. 82:1974-1978 (1985); Guild andStrominger, J. Biol. Chem. 259:9235-9240 and 13504-13510 (1984). Asimilar site is found in the intracytoplasmic domain of the interleukin2 receptor, and is phosphorylated in vivo by protein kinase C. Leonardet al., Nature 311:626-631 (1984); Nikaido et al., Nature 311:631-635(1984); Shackelford and Trowbridge, (1984) J. Biol. Chem. 259:11706.

Immunoprecipitation of CD2 antigen expressed by transfected cells

COS cells were transfected with the CD2 expression plasmid and surfacelabeled with ¹²⁵ I by the lactoperoxidase method 60 hourspost-transfection. A cell lysate was prepared, and portions wereincubated either with monoclonal anti-CD2 antibody (OKT11) or with anextraneous (OKT4; anti-CD4) antibody for 2 hours at 4° C.Sepharose-bound anti-mouse antibody was added, and after several washingsteps, the adsorbed proteins were eluted and electrophoresed through a11.25% acrylamide gel together with similarly preparedimmunoprecipitates from phytohemagglutinin-activated T lymphocytes, thecDNA donor line HPB-ALL, or a long-term T cell line generated in thislaboratory. Autoradiography demonstrated a prominent band ofimmunoreactive material precipitated from transfected COS cells by theanti-CD2 antibody, but not by the control. The calculated mean molecularweight of the COS cell material was 51 kd, compared to a mean molecularweight of 54 kd for the T blast and T cell line material; the antigenfrom HPB-ALL cells was found to have a molecular weight of approximately61 kd. The observed differences in size were attributed to differentpatterns of glycosylation in the different cell types. A minor band ofapparent molecular weight 38 kd was present in materialimmunoprecipitated from COS cells but not from T cells or HPB-ALL cells.The size of this species agrees within experimental error with thepredicted molecular weight of mature unglycosylated peptide, 39 kd.

COS cells expressing CD2 form rosettes with sheep erythrocytes

COS cells transfected with the CD2 expression clone were treated for 1hour with purified MT910 (IgG, kappa) anti-CD2 antibody (Rieber et al.,Leukocyte Typing II, Vol. I, pp. 233-242 (1986)) at a concentration of 1μg/ml, or with purified MB40.5 (IgGl, kappa; Kawata et al., J. Exp. Med.160:633-651 (1984)) antibody at the same concentration. MB40.5recognizes a monomorphic HLA-ABC determinant and cross-reacts withAfrican Green Monkey histocompatibility antigens; it was chosen becauseit represents an isotype-matched antibody recognizing a surface antigenof approximately the same abundance as the CD2 antigen expressed bytransfected cells. Sheep erythrocyte rosettes were observed in thepresence of MB40.5, but not of MT910. Rosette inhibition was alsoobserved with OKT11 antibody, and not with various other controlantibodies.

Transfected COS cells form rosettes with other animal erythrocytes

In addition to sheep erythrocytes, human T cells are known to formrosettes with horse, pig, dog, goat, and rabbit, but not mouse or raterythrocytes. Johansen et al., J. Allergy Clin. Immunol. 54:86-94(1974); Amiot et al., in, A. Bernard et al., eds., Leucocyte Typing,Springer, publisher, New York, N.Y., pp. 281-293 (1984); Nalet andFournier, Cell. Immunol. 96:126-136 (1985). Autorosettes between humanerythrocytes and human thymocytes (Baxley et al., Clin. Exp. Immunol.15:385-393 (1973)) have also been reported. COS cells transfected withthe CD2 expression clone were treated with either MT910 or with thecontrol antibody, MB40.5, and exposed to erythrocytes from the speciesabove. Rosettes were observed with horse, pig, dog, goat, sheep, rabbit,and human erythrocytes, but not with mouse or rat erythrocytes. Rosetteformation was blocked by pretreatment of transfected COS cells withMT910, but not with MB40.5. In these experiments, it was noticed thathorse erythrocytes formed unusually dense rosettes, and that goaterythrocytes formed rather sparse rosettes, possibly because their smallsize made them more susceptible to washing. Mouse erythrocytes showedweak spontaneous binding to the culture dish as well as to MT910 andMB40.5 pretreated cells, while rat erythrocytes showed no detectablebinding of any sort.

Binding of human erythrocytes is blocked by LFA3 antibody

Because it has been suggested on the basis of antibody blocking studiesthat LFA3 is the target structure for the CD2 antigen (Shaw et al.,Nature 323:262-264 (1986)), the ability of anti-LFA3 antibody to preventrosette formation was investigated. Transfected cells were exposed tohuman erythrocytes pretreated for 2 hours with either anti-LFA3 (IgGl,kappa) as ascites at 1:1000 dilution, or with a 10 μg/ml concentrationof each of four isotype-matched nonagglutinating antibodies directedagainst human erythrocyte antigens as prevalent or more prevalent thanLFA3:G10/B11 and D10, anti-K14 antigen, D6, anti-Wr^(b) antigen; andF7/B9, anti-k antigen. Nichols et al., Vox Sang, in press. Theerythrocytes were washed free of excess LFA3 antibody, but were allowedto form rosettes in the presence of the control antibodies to guardagainst possible loss of antibody blocking power by desorption. Rosetteformation was observed in the presence of all four control antibodies,but not with erythrocytes pretreated with anti-LFA3.

COS cells expressing other T cell antigens do not form rosettes

A number of clones were isolated by the same expression technique usedto clone CD2 and characterized to varying degrees by antibodyreactivity, nucleic acid restriction and sequence analysis, andimmunoprecipitation. Representative clones were transfected into COScells and analyzed for ability to sustain rosette formation. The CD1a,CD1b, CD1c, CD4, CD5, CD6, CD6, CD8, and CD28 (Tp44) clones did not formrosettes with human erythrocytes.

RNA blot analysis

Equal amounts of total RNA prepared from cell types expressing orlacking CD2 antigen were electrophoresed through denaturing agarose gelsand transferred to nylon. Hybridization of the transferred RNA with astrand selective probe (Hu and Messing, Gene 17:271-277 (1982)) preparedfrom an M13 clone containing a CD2 cDNA insert revealed the presence ofprominent 1.65 and 1.3 kb transcripts present in RNA derived fromthymocyte, activated T cell, and senescent T cell populations. Lesseramounts were found in RNA extracted from the cDNA donor line, HPB-ALLand less still from MOLT4; barely detectable levels were recorded in RNAfrom the HSB-2 line. No reactivity was observed with RNA from Namalwa(Burkitt lymphoma), U937 (histiocytic leukemia), HuT-78 (Adult T cellleukemia), PEER (T cell leukemia), or Jurkat clone J3R7 (T cellleukemia) lines. The pattern of reactivity conformed well with the knownor measured pattern of expression of CD2 antigen, which was absent orindetectable on the Namalwa, *937, HuT-78, J3R7, PEER, and HSB-2 celllines, weakly present on MOLT4, more strongly present on EPB-ALL, andmost strongly present on activated T cells. Thymocytes are also known toexpress high levels of CD2 antigen.

Examination of the sequence of the cDNA clone suggested that the 1.3 kbRNA might arise by formation of an alternate 3' end distal to thecanonical polyadenylation signal AATAAA at position 1085 in the cDNAsequence. To test this notion, RNA from HPB-ALL and activated T cellswas subjected to Northern blot analysis and hybridized either with acomplete cDNA probe, or with a probe derived from the 3' portion of thecDNA distal to nucleotide 1131. The latter probe reacted only with the1.65 kb species, while the former showed the same reactivity patternobserved in FIG. 5. This result is consistent with the suggested originof the 1.3 kb transcript.

In both activated and senescent T cell RNA preparations, a weaklyhybridizing transcript of approximately 0.75 kb was detected. At presentthe origin of this RNA is unknown.

Genomic organization of the CD2 gene

Southern blot analysis of genomic DNA from placenta, peripheral bloodlymphocytes, T cells, HeLa cells, or the tumor lines used in the RNAanalysis above showed identical BamHI digest patterns, indicating thatrearrangement is not involved in the normal expression of the CD2 geneduring development. Similarly, no gross genomic alteration underlies thefailure of the examined T cell tumor lines to express CD2 antigen.Restriction analysis of total genomic DNA with a number of otherenzymes, as well as preliminary results with an incomplete collection of1 phage recombinants bearing the CD2 sequence, shows that the gene isdivided into at least four exons.

EXAMPLE II Isolation and Molecular Cloning of Human LFA-3 Antigen

The previous example shows that cDNAs encoding surface antigens, such asthe CD2 antigen, can be isolated by the transient expression system ofthe present invention, in which COS cells transfected with cDNAlibraries are allowed to attach to ("panned" on) antibody-coated plates.Plasmid DNA is recovered from cells adhering to the plates, transformedinto E. coli, and the process is repeated, usually twice, to isolate thedesired clone. Although powerful, this approach cannot be used when themonoclonal antibodies used for panning recognize determinants on theuntransfected cells. This appears to be the case for anti-LFA3monoclonal TS2/9. However, a similar transient expression system basedon polyoma virus replication-competent cells should allow almost allmonoclonals to be used, since the probability of cross reaction betweenmurine antibodies and murine cell surface determinants should usually besmall.

A new expression vector, CDM8 (FIG. 3) was created from the COS cellvector piH3M described previously. The new vector differs by theinclusion of a deleted version of a mutant polyoma virus early regionselected for high efficiency expression in both murine and monkey cells,by the replacement of substantially all of the human immunodeficiencypromoter region with the cognate sequences of the human cytomegalovirusimmediate early promoter, and by inclusion of a bacteriophage T7promoter between the eukaryotic promoter and the site of cDNA insertion.Expression in COS cells of chloramphenicol acetyltransferase by all ofthe vectors was equivalent.

A library of 1.9×10⁶ recombinants having inserts greater than 0.8 kb insize was prepared in the CDM8 vector from a microgram of poly A⁺ RNAisolated from the human lymphoblastoid cell line JY. The library wasintroduced into WOP cells (NIH 3T3 cells transfected with polyoma origindeletion DNA) by spheroplast fusion, and subjected to three rounds ofpanning and reintroduction into E. coli as described in Example I.

A clone encoding the LFA-3 antigen was identified by indirectimmunofluorescence of transfected WOP cells, amplified and sequenced(FIG. 4A). Within the 874 bp insert, an open reading frame of 237residues originates at a methionine codon closely corresponding to theconsensus sequence suggested by Kozak, Microbiol. Rev. 47:1-45 (1983).The reading frame terminates in a series of hydrophobic residues lackingthe characteristic basic anchoring residues of internal membraneproteins, but sharing features with known phosphatidylinositol-linkedsuperficial membrane proteins. The features include clustered serine orthreonine residues in a hydrophilic region immediately preceding thehydrophobic domain, and the presence of serines and threonines in thehydrophobic portion.

The amino acid sequence predicted from the nucleotide sequence of theLFA-3 clone was compared to the NBRF database, and no significanthomologies were uncovered; the most significant scores were to the HIVenvelope protein. Within the 200 residues comprising the presumed matureprotein are 6 N-linked glycosylation sites, and 5 tandem serine ortandem threonine residues that frequently appear in O-linkedglycosylated proteins. Ten cysteine residues appear in the completesequence, 6 of which are distributed in the latter half of the matureprotein, and one of which falls in the carboxy-terminal hydrophobicdomain. Although esterification of cysteine thiols to fatty acids is acommon occurrence in integral membrane proteins, and may play analternate role in membrane anchoring of LFA-3, two examples are known ofcysteine residues within or at the margin of the hydrophobic region ofphosphatidylinositol linked proteins.

The predicted sequence suggests that the known manipulations forincreasing erythrocyte adhesion to T cells may find direct physicalexplanation in the structure of the LFA-3 molecule.Aminoethylisothiouronium bromide, the thiourea adduct ofbromoethylamine, undergoes spontaneous rearrangement tomercaptoethylguanidine at alkaline pH. The latter likely gains access todisulfide bonds inaccessible to less chaotropic reducing agents and maythereby reduce and promote the unfolding of the LFA-3 molecule.Similarly, neuraminidase may decrease steric interference by the manycarbohydrate chains on the molecule.

RNA and DNA blot hybridization analysis showed that the LFA-3 geneshares no closely related sequences in the genome, and encodes a singleRNA species of about 1 kb in length. Cell lines that express largeamounts of surface LFA-3 have greater amounts of LFA-3 RNA than thosethat express small or nondetectable amounts.

Radioimmunoprecipitation of the antigen expressed in transfected COS andmurine cells shows a broad band of approximately 50 kd mean molecularmass, similar to that found in JY cells.

EXAMPLE III Isolation and Molecular Cloning of the Human CD28 cDNAAntigen

The previous examples illustrate the monoclonal antibody-based techniqueof the present invention for enrichment of cDNAs encoding surfaceantigens. In the present example, a method of constructing plasmidexpression libraries is described which allows the enrichment techniqueto be fully exploited. The method of the present invention for makingplasmid expression libraries is of general use for expression cloning.

The antibody selection technique of the present invention has also beenapplied to isolate a cDNA clone encoding the CD28 antigen. The antigenshares substantial homology with members of the immunoglobulinsuperfamily and forms a dimer structure on the surface of transfectedCOS cells similar to the dimer structure found on T lymphocytes.

Preparation of cDNA Libraries

Poly(A)+ RNA was prepared from the human T-cell tumor line HPB-ALL byoligo(dT) cellulose chromatography of total RNA isolated by theguanidinium thiocyanate method (Chirgwin, J. M. et al., Biochemistry18:5294-5299 (1979)). cDNA was prepared by a protocol based on themethod of Gubler and Hoffman (Gubler, U. et al., Gene 25:263-269(1982)). 4 μg of mRNA was heated to approximately 100° C. in a 1.5 mlcentrifuge tube for 30 seconds, quenched on ice, and the volume adjustedto 70 μl with RNAse-free water. To this were added 20 μl of buffer(0.25M Tris pH 8.8 (8.2 at 42° C.), 0.25M KCl, 30 mM MgCl₂), 2 μl ofRNAse inhibitor (Boehringer 36 U/μl), 1 μl of 1M DTT, 1 μl of 5 μg/μl ofoligo dT (Collaborative Research), 2 μl of 25 mM each deoxynucleosidetriphosphate (U.S. Biochemicals), and 4 μl of reverse transcriptase(Life Sciences, 24 U/μl). After 40 minutes at 42° C., the reaction wasterminated by heating to 70° C. for 10 minutes. To the reaction mix wasthen added 320 μl of RNAse free water, 80 μl of buffer (0.1M Tris pH7.5, 25 mM MgCl₂, 0.5M KCl, 0.25 mg/ml BSA, and 50 mM DTT), 25 units ofDNA Polymerase I (Boehringer), and 4 units of RNAse H (BRL). After 1hour at 15° C. and 1 hour at 22° C., 20 μl of 0.5M EDTA pH 8.0 wereadded, the reaction mixture was extracted with phenol, NaCl was added to0.5M, linear polyacrylamide (carrier; Strauss, F. et al., Cell37:889-901 (1984)) was added to 20 μg/ml, and the tube was filled withethanol. After centrifugation for 2-3 minutes at 12,000×g, the tube wasremoved, vortexed to dislodge precipitate spread on the wall of thetube, and respun for 1 minute.

Unpurified oligonucleotides having the sequence CTCTAAAG andCTTTAGAGCACA were dissolved at a concentration of 1 mg/ml, MgSO₄ wasadded to 10 mM, and the DNA precipitated by adding 5 volumes of EtOH.The pellet was rinsed with 70% ETOH and resuspended in TE at aconcentration of 1 mg/ml. 25 μl of the resuspended oligonucleotides werephosphorylated by the addition of 3 μl of buffer (0.5M Tris pH 7.5, 10mM ATP, 20 mM DTT, mM spermidine, 1 mg/ml BSA, and 10 mM MgCl₂) and 20units of polynucleotide kinase followed by incubation at 37° C.overnight.

3 μl of the 12-mer and 2 μl of the 8-mer phosphorylated oligonucleotideswere added to the cDNA prepared as above in a 300 μl reaction mixturecontaining 6 mM Tris pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 0.35 mg/ml BSA, 7 mMmercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine and 400 units T4DNA ligase (New England BioLabs) at 15° overnight. 10 μl of 0.5M EDTAwere added, the reaction was phenol extracted, ethanol precipitated,resuspended in a volume of 100 μl and layered on a 5 ml gradient of5-20% potassium acetate in 1 mM EDTA, 1 μg/ml ethidium bromide. Thegradient was spun 3 hours at 50,000 rpm (SW55 rotor) and fractionatedmanually, collecting three approximately 0.5 ml fractions followed bysix approximately 0.25 ml fractions in microcentrifuge tubes by means ofa butterfly infusion set inserted just above the curve of the tube.Linear polyacrylamide was added to 20 μg/ml, the tubes were filled withethanol, chilled, spun, vortexed and respun as above. The precipitatewas washed with 70% ethanol, dried, and resuspended in 10 μl. 1 μl ofthe last 6 fractions was run on a gel to determine which fractions topool, and material less than 1 kb in size was typically discarded.Remaining fractions were pooled and ligated to the vector.

The complete sequence and derivation of the vector is shown in FIG. 5.The vector was prepared for cloning by digestion with BstXI andfractionation on 5-20% potassium acetate gradients as described for thecDNA. The appropriate band was collected by syringe under 300 nm UVlight and ethanol precipitated as above. cDNA and vector were titratedin test ligations. Usually 1-2 μg of purified vector were used for thecDNA from 4 μg of poly A+ RNA. The ligation reactions were composed asdescribed for the adaptor addition above. The ligation reactions weretransformed into MC1061p3 cells made competent as described above. Thetransformation efficiency for supercoiled vector was 3-5×10⁸colonies/μg.

Recovery and characterization of the CD28 clone

Panning of the library was carried out as described herein above, usingpurified antibody 9.3 (DuPont) at a concentration of 1 ug/ml in theantibody cocktail. The methods used for COS cell transfection,radioimmunoprecipitation, RNA and DNA blot hybridization, and DNAsequencing were all as described herein above.

To isolate the CD28 cDNA, a large plasmid cDNA library was constructedin a high efficiency expression vector containing an SV40 origin ofreplication. A preferred version of the vector, containing an M13origin, is shown in FIGS. 6A-6B. Three features of the vector make itparticularly suitable for this use: (i) the eukaryotic transcriptionunit allows high level expression in COS cells of coding sequencesplaced under its control; (ii) The small size and particular arrangementof sequences in the plasmid permit high level replication in COS cells;and (iii) the presence of two identical BstXI sites in invertedorientation and separated by a short replaceable fragment allows the useof an efficient oligonucleotide-based strategy to promote cDNA insertionin the vector.

The BstXI cleavage site, CCAN'₅ NTGG, creates a four base 3' extensionwhich varies from site to site. A vector was created in which twoidentical sites were placed in inverted orientation with respect to eachother, and separated by a short replaceable segment of DNA. Digestionwith BstXI followed by removal of the replaceable segment yielded avector molecule capable of ligating to fragments having the same ends asthe replaceable segment, but not to itself. In parallel, cDNA syntheticoligonucleotides were employed that give the same termini as thereplaceable segment. The cDNA then could not ligate to itself, but couldligate to the vector. In this way, both cDNA and vector were used asefficiently as possible.

Tailing with terminal transferase achieves the same end, but with lessconvenience and less overall efficiency. Moreover, homopolymer tractslocated 5' to cDNA inserts have been reported to inhibit expression invitro and in vivo (Yokota, T., et al., Nucl. Acids Res. 14:1511-1524(1986); Riedel, H., EMBO J. 3:1477-1483 (1985)). Similar approachesbased on the use of partially filled restriction sites to favorinsertion of genomic DNAs (Zabarovsky, E. R., et al., Gene 42:119-123(1986)) and cDNAs (Yang, Y., et al., Cell 47:3-10 (1986)) recently havebeen reported. These approaches give 2 or 3 base complementary termini,which usually ligate less efficiently than the 4 base extensionsreported here.

Although the cloning scheme of the present invention does not result ina directional insertion of the cDNA, the ability to make large librarieseasily, coupled with a powerful selection procedure, makes directionalinsertion unnecessary. The library construction efficiencies observedaccording to the present invention, between 0.5 and 2×10⁶ recombinantsper μg of mRNA, with less than 1% background and an insert size greaterthan 1 kb, compared favorably with those described for phage vectorslambda gt10 (7.5×10⁵ /μg of mRNA) and lambda gt11 (1.5×10⁶ /μg of mRNA)(Huynh, T., et al., In: DNA Cloning Vol. I, A Practical Approach,Glover, D. M. (ed.), IRL Press, Oxford (1985), pp. 49-78); but theresulting clones were more convenient to manipulate.

Surface antigen cDNAs can be isolated from these libraries using theantibody enrichment method of the present invention. In this method, thelibrary is introduced into COS cells (for example, by spheroplast orprotoplast fusion), where it replicates and expresses its inserts. Thecells are harvested by detaching without trypsin, treated withmonoclonal antibodies specific for the surface antigens desired, anddistributed in dishes coated with affinity purified antibody to mouseimmunoglobulins. Cells expressing surface antigen adhere, and theremaining cells can be washed away. From the adherent cells, a Hirtfraction is prepared (Hirt, B., J. Molec. Biol. 26:365-369 (1967)), andthe resulting DNA transformed back into E. coli for further rounds offusion and selection. Typically, after two rounds of selection withmonoclonal antibodies recognizing different surface antigens, a singleround of selection is performed with a single antibody, or pool ofantibodies recognizing the same antigen.

Isolation of a CD28 cDNA

The CD28 cDNA was isolated from a library of about 3×10⁵ recombinantsprepared from cDNA from 0.8 μg of poly A⁺ RNA using an earlier versionof the protocol described in the Materials and Methods. The library wasscreened for CD28 (and other surface antigen) cDNA clones by the methodoutlined above. After the third transfection, COS cells were panned withthe 9.3 antibody alone. A Hirt supernatant was prepared from theadherent cells and transformed into E. coli. Plasmid DNA was isolatedfrom eight colonies and transfected individually into COS cell cultures.The presence of the CD28 antigen was detected in three of eighttransfected cultures by indirect immunofluorescence. All three plasmidDNAs contained an insert of about 1.5 kb.

cDNA sequence analysis

The CD28 cDNA encodes a long open reading frame of 220 residues havingthe typical features of an integral membrane protein (FIGS. 7A-7B).Removal of a predicted (von Heijne, Nucl. Acids Res. 14:4683-4690(1986)) N-terminal signal sequence gives a mature protein of 202residues comprising an extracellular domain with five potential N-linkedglycosylation sites (Asn-X-Ser/Thr), a 27-amino acid hydrophobicmembrane spanning domain, and a 41-amino acid cytoplasmic domain.Comparison of the amino acid sequence of CD28 with the NationalBiomedical Research Foundation database (Version 10.0) revealedsubstantial homology with mouse and rabbit immunoglobulin heavychainvariable regions over a domain spanning almost the entire extracellularportion of CD28. Within this domain two cysteine residues in thehomology blocks Leu-(Ser or Thr)-Cys and Tyr-(Tyr or Phe)-Cys are sharedby CD28, CD4, CD8, immunoglobulin heavy- and light-chain variablesequences and related molecules with approximately the same spacing(Maddon et al., Annu. Rev. Biochem. 48:961-997 (1979)).

CD28 cDNA directs the production of a homodimer in transfected COS cells

Immunoprecipitation of CD28 antigen from transfected COS cells wascarried out using the monoclonal antibody 9.3 (Hansen, J. A., et al.,Immunogenetics 10:247-260 (1980)). The material obtained from COS cellsmigrated with a molecular weight of 74 kd under nonreducing conditionsand 39 kd under reducing conditions, a pattern consistent with homodimerformation. Under the same conditions activated T cells give bands withmolecular weights of 87 and 44 kd, and HPB-ALL cells give bands of 92and 50 kd, under nonreducing and reducing conditions respectively. Thevariation in molecular weight of the material obtained from differentcell types arises as a result of differing glycosylation patternscharacteristic of each type. Similar results were observed with otherleukocyte surface antigens (Seed et al., Proc. Natl. Acad. Sci USA 87(1987)). The nucleotide sequence of the CD28 cDNA predicts a matureprotein with molecule weight of 23 kd, much smaller than observed inthese experiments, and probably attributable to utilization of the 5N-linked glycosylation sites predicted by the amino acid sequence.

RNA blot analysis

Equal amounts of total RNA prepared from cell types expressing orlacking CD28 were subjected to RNA blot analysis as describedhereinabove. Four bands with molecular weights of 3.7, 3.5, 1.5, and 1.3kb were visible in lanes containing RNA thymocytes, T blasts, senescentT cells, and the T cell leukemia cell lines PEER and HPB-ALL. No bandswere detected in lanes containing RNA prepared from the cell lines U937(histiocytic leukemia), HuT-78 (Adult T cell leukemia), Jurkat (T cellleukemia), Namalwa (Burkitt lymphoma), MOLT4, and HSB-2, all of which donot express CD28. The 1.5 kb transcript presumably corresponds to theisolated cDNA, and the 3.7 and 3.5 kb species reflect incompletesplicing or alternative polyadenylation site utilization. The 1.3 kbtranscript may terminate at an unconventional polyadenylation signal,since there is no obvious candidate in the sequence.

The CD28 gene is not rearranged

DNA blot analysis (Seed et al., Proc. Natl. Acad. Sci USA 87 (1987)) ofgenomic DNA from placenta, peripheral blood lymphocytes, T cells, HeLacells, or the tumor lines used in the RNA blot analysis above showedidentical Dra 1 digest patterns indicating that rearrangement is notinvolved in the normal expression of the CD28 gene during development.Similarly, no gross genomic rearrangement underlies the failure of theexamined T-cell tumor lines to express CD28 antigen. It may be inferredfrom the Dra 1 fragment pattern that the CD28 gene contains at least twointrons.

EXAMPLE IV Isolation and Molecular Cloning of Two Human CD7 AntigencDNAs

The CD7 cluster of antibodies (Palker, et al., Leukocyte Typing II,Springer-verlag, New York, 303-313 (1985)) recognized a 40 kdglycoprotein (gp40) on the surface of peripheral blood T cells andthymocytes. Early studies with anti-CD7⁺ antibodies showed that CD7+Tcells enhance immunoglobulin (Ig) synthesis by B cells (Miroshima etal., J. Immunol. 129:1091-1098 1982)), suppress B cell Ig synthesis whenstimulated with Concanavalin A (Haynes et al., Proc. Natl. Acad. Sci.U.S.A. 76:5829-5833 (1979)) and are the precursors of the cytotoxic Tcells generated in mixed lymphocytic culture (Morishima et al., J.Immunol. 129:1091-1098 (1982)). Furthermore, CD7 has been found to bethe most reliable marker for the identification of T cell acutelymphoblastic leukemia (Link et al., Blood 62:722-728 (1983)). As such,studies have been carried out, in which cytotoxins coupled to theanti-CD7 antibody 3A1 were used to purge bone marrow prior to reinfusionto avoid early relapse in autologous bone marrow transplants or asprophylaxis against graft vs. host disease in allogenic bone marrowtransplants (Ramakrishnan et al., J. Immuol. 135:3616-3622 (1985)).Similarly, anti-CD7 antibodies also show promise as immunosuppressiveagents in the treatment of allograft rejections (Raftery et al.,Transpl. Proc. 17:2737-2739 (1985)) which is in accord with the recentobservation that the anti-CD7 antibody 7G5 significantly inhibits theprimary mixed lymphocyte reaction (Lazarovits et al., Leukocyte TypingIII. Oxford Univ. Press, Oxford (1987)).

At present the physiological role of CD7 is not understood. It is knownthat anti-CD7 antibodies are not mitogenic, and do not block the Tcells' response to PHA, or tetanus toxoid (Palker et al., LeukocyteTyping, Springer-Verlag, New York, 303-313 (1985)). Some have noted thatexpression of CD7 in thymocytes occurs prior to the onset of T cellreceptor beta-chain rearrangement (Pittaluga et al., Blood 68:134-139(1986)) and have pointed to a possible role for CD7 in thisrearrangement and subsequent expression of the T cell receptor. It isclear that the cloning of the CD7 antigen would further efforts tounderstand its role in T cell physiology. Nucleotide sequencing andpreliminary characterization of two cDNAs encoding the CD7 antigen wascarried out according to the method of the present invention. Promptedby the recent suggestion that CD7 may be, or be part of, the T cell IgMreceptor (Sandrin et al., Leukocyte Typing III. Oxford Univ. Press,Oxford (1987)), the ability of COS cells expressing CD7 to bind IgM orIgM immune complexes was evaluated. The results do not support thesimple notion that CD7 itself is an IgM receptor.

Preparation of cDNA library and recovery and characterization of CD7clones

Preparation of an HPB-ALL cDNA library in the expression vector piH3 wascarried out as described herein. Panning of the library was carried outaccording to the method of the present invention, using purifiedanti-CD7 antibody Leu9 (Becton Dickinson) and antibody 7G5 as ascitesfluid was diluted 1/1000. Methods for cell transfection,radioimmunoprecipitation, DNA and RNA blot hybridization and DNAsequencing were all as described herein.

IgM and IgG binding by COS cells transfected with CD7 and CDw32

Human IgM, IgG, and IgA antibodies, affinity purified FITC conjugatedgoat anti-human immunoglobulins antibodies (anti-Ig(G+M+A)), washed andpreserved bovine red blood cells, and IgG and IgM fractions of rabbitanti-bovine red blood cell antibodies were purchased from CooperBiomedical (Malverne, Pa.). COS cells were transfected by the DEAEDextran method with cDNAs encoding the CD7, CDw32, and CD28 surfaceantigens. 48 hours after transfection the cells were washed withPBS/0.5% BSA and incubated with either human IgM, IgG or IgA antibodiesat a concentration of 1 μg/ml, at 4° C. for 2 hours. Subsequently thecells were washed with PBS/0.5% BSA and incubated for 30 minutes at 4°C. with FITC conjugated rabbit anti-human immunoglobulins. After washingthe cells were examined with a fluorescence microscope. The experimentswere also performed in the presence of 0.1% azide with the same results.

Bovine erythrocytes for rosette assays were prepared as described byErcolani et al., J. Immunol. 127:2044-2051 (1981). Briefly, a 2%suspension of bovine erythrocytes was washed with PBS/0.5% BSA andtreated with subagglutinating amounts of either IgG or the IgM fractionof rabbit anti-bovine erythrocyte antibodies at 4° C. for 1 hour.Erythrocytes were then washed twice with PBS/0.5 BSA and adjusted to a2% solution. 2 ml of antibody-coated erythrocytes were layered on 60 mmdishes containing COS cells which had been transfected 48 hours earlierwith either CD7, CD32 or CD28 by the DEAE Dextran method. The disheswere then centrifuged at 150×g at 4° C. for 15 minutes. After anadditional 45 minute incubation at 4° C., the plates were gently washed5 times with 5 mls of PBS/0.50 BSA, and the COS cells were examined forrosette formation. These experiments were also performed in the presenceof 0.1% sodium azide without alteration of the results.

Formation of T cell rosettes with antibody-coated erythrocytes

Peripheral blood lymphocytes were obtained from heparinized blood bycentrifugation at 4° C. over a Ficoll-Hypaque gradient at 400×g for 30minutes. Leukocytes at the interface were washed two times with PBS. Theleukocytes were adjusted to 10Y7 cells/ml in IMDM/10% Fetal Bovine Serum(FBS) and incubated in tissue culture dishes at 37° C. for 30 minutes.Nonadherent cells were transferred to new dishes, and PHA was added tostimulate proliferation of T lymphocytes. On the next day the cells werewashed with PBS and placed in fresh IMDM/10% FBS.

Rosette assays were performed three days later. Cells were washed withPBS/0.5% BSA, and a 10 ul suspension of 2% Ig-coated erythrocytesprepared as described above was added to 10 ul of PBS/0.5% BSAcontaining 5×10⁶ cells/ml. The mixtures were placed in Falcon roundbottom 96 well plates and centrifuged at 150×g for 15 min at 4° C. Afteran additional incubation of 45 min at 4° C. pellets were resuspendedwith 10 ul of PBS/0.5% BSA, and the rosettes scored by phase contrastmicroscopy. The experiments were carried out in both the presence andabsence of 0.1% sodium azide with no detectable difference.

Isolation of cDNAs encoding the human CD7 antigen

To isolate CD7 cDNAs, a large plasmid library was constructed in theexpression vector piH3M as describe hereinabove. The library wasintroduced into COS cells by spheroplast fusion, and allowed toreplicate and express its inserts. The COS cells were harvested bydetaching without trypsin 48 to 72 hours after transfection, treatedwith monoclonal antibodies specific for surface antigens believed to beencoded in the library, and distributed in dishes coated with affinitypurified anti-mouse antibody as described herein. Under theseconditions, cells expressing surface antigen adhere and the remainingcells can be washed way.

A Hirt (Hirt, J. Mol. Biol. 26:365-369 (1967)) fraction was preparedfrom adherent cells, and the resulting DNA transformed back into E. colifor further rounds of fusion and selection. In the third round ofselection the detached cells were treated with a mixture of monoclonalantibodies specific for CD7 (765 and Leu9), and a Hirt supernatant wasagain generated and transformed into E. coli . After transformation ofthe DNA into E. coli 8 colonies were picked, and the plasmid DNAprepared from them by an alkaline miniprep procedure (Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1982)). DNA was prepared from 8 resulting coloniesand transfected into COS cells. After 3 days, surface expression of theCD7 antigen was detected by indirect immunofluorescence in 7 of 8transfected dishes. Restriction enzyme digest of the correspondingplasmid DNAs revealed two species. One contained a 1.2 kb insert, andthe other a 1.3 kb insert.

CD7 cDNA sequence analysis

Both isolates were sequenced by the dideoxynucleotide method. The 1.2 kbcDNA encodes a long open reading frame of 240 residues having thetypical features of an integral membrane protein. The initial assignmentof the signal sequence cleavage site by the method of von Heijne (Nucl.Acids Res. 14:4683-4690 (1986)) was at the 18th residue. It later wasdetermined, however, that the homology with immunoglobulin variableregions would better predict the mature terminus at residue 26; thisassignment would also correlate well with the position of the intron asdiscussed below and as shown in FIG. 8. Removal of the predictedN-terminal signal sequence gives a mature protein of 215 residues with apredicted molecular mass of 23 kd. In the extracellular domain are twoN-linked glycosylation sites (Asn-X-Ser Thr), in agreement with theresults of Sutherland et al. (J. Immunol. 133:327-333 (1984)), who alsoshowed the presence of O-linked glycans and covalently associatedpalmitic acid on the mature protein. In the 27 amino acid hydrophobicmembrane spanning domain is a single cysteine residue which may be thesite of fatty acylation (Rose et al., Proc. Natl. Acad. Sci. USA81:2050-2054 (1984); Kaufman et al., J. Biol. Chem. 259:7230-7238(1984)). The length of the cytoplasmic domain, 39 residues, is in goodagreement with the 30-40 amino acids predicted by protease digestion ofthe CD7 precursor in rough microsomal membrane fractions (Sutherland etal., J. Immunol. 133:327-333 (1984)).

Sequence analysis of the 1.7 kb clone (FIG. 8) revealed the presence ofan intron located 121 bp from the 5' end. The 411 bp intron containsstop codons in all three reading frames and is located just downstreamof the secretory signal sequence, as is frequently observed for secretedor surface proteins. Both the 5' and 3' ends of the intron conform tothe splice donor/acceptor consensus AAG GTRAGA/ . . . /Y₆₋₁₁ NYAG A(Mount, Nucl. Acids Res. 10:459-472 (1982)). Because both the 1.2 and1.7 kb clones express CD7 antigen equally well in COS cells, the intronmust be excised in COS cells fairly efficiently.

Comparison of the amino acid sequence with the National BiomedicalResearch Foundation database revealed substantial homology with humanand mouse immunoglobulin kappa chain and T-cell receptor gamma chainvariable regions over almost the entire extracellular portion of themolecule. Two cysteine residues shared in approximately equal spacing byall three structures fall in the conserved sequences Ile-Thr-Cys andTyr-X-Cys. In kappa chain variable regions these cysteines form adisulfide bridge. The presence of at least one intrastrand disulfidebond in the CD7 structure has previously been proposed by Sutherland etal. (J. Immunol. 133:327-333 (1984) ), who noted thatimmunoprecipitation of CD7 gave rise to a band with an apparentmolecular mass of 40 kd under reducing conditions and 38 kd undernonreducing conditions.

Based on the homology with immunoglobulin V-regions, it is predictedthat CD7 contains a disulfide bond linking Cys 23 and Cys 89. A seconddisulfide bond, linking Cys 10 and Cys 117, has been proposed, based onthe structural similarity between CD7 and Thy-1. The extracellulardomains of both Thy-1 and CD7 have 4 cysteine residues, in roughlyhomologous positions. The 4 cysteine residues of Thy-1 are joined in twointernal disulfide bridges between Cys 9-111 and Cys 19-85 (Williams etal., Science 216:696-703 (1982)). In Thy-1, Cys 111 forms an amide bondwith the ethanolamine moiety of a substituted phosphatidylinositol, andis thus the last residue of the mature molecule (Tse et al., Science230:1003-1008 (985) ). In CD7, Cys 117 is followed by four repeats of asequence whose consensus is Xaa-Pro-Pro-Xaa-Ala-Ser-Ala-Leu-Pro, andwhich, it is proposed, plays the role of a stalk projecting the V-likedomain away from the surface of the cell.

In addition to the homologies shown in FIG. 20 and mentioned above, theextracellular domain of CD7 has significant homology with both chains ofthe rat CD8 heterodimer (Johnson et al., Nature 323:74-76 (1986)), andthe myelin P₀ protein (Lemke et al., Cell 40:501-508 (1985)).

CD7 directs the production of a 40 kd protein in transfected COS cells

Immunoprecipitation of CD7 antigen from transfected COS cells wascarried out as described herein using monoclonal antibody 7G5(Lazarovits et al., Leukocyte Typing III, Oxford Univ. Press, publisher,Oxford, England (1987). The material obtained from COS cells migratedwith as a broad band with molecular weight of 40 kd under reducingconditions. Under the same conditions HPB-ALL cells (the cDNA donorline) and activated T cells gave bands with molecular widths of 41 and39 kd respectively. In both the COS cell and HPB-ALL lane a faint bandwith molecular weight of 30 kd was also observed, possibly correspondingto a partially glycosylated precursor (Sutherland, D. R., et al., J.Immunol. 133:327-333 (1984)).

RNA blot analysis

Equal amounts of total RNA prepared from cell types expressing orlacking CD7 were subjected to Northern blot analysis as describedherein. A single 1.3 kb species was visible in lanes containing RNA fromthymocytes, activated T cells, resting T cells, and the T cell leukemialines HuT-78, HPB-ALL, Jurkat J3R7, HSB-2 and PEER. With the exceptionof the PEER cell line, none of the T cell tumors showed significantoverexpression of CD7 transcripts. CD7 RNA was detected in all of thethymus-derived cells, but not in RNA from U937 (histiocytic leukemia)and Namalwa (Burkitt Lymphoma) cells. No band corresponding to the 1.7kb cDNA could be detected, suggesting that this species is artificiallyenriched during the cloning or library amplification process.

Enrichment during amplification seems unlikely because the 12 kb cDNAclone propagates as well in E. coli as the 1.7 kb clone. However,immediately upstream and downstream from the site of insertion of theintron are sequences that could form an interrupted stem and loopstructure. Eight of the 10 basepairs of the potential stem are GC pairs,perhaps giving the structure sufficient stability to interfere withelongation of the cDNA first strand. The presence of the intron greatlyseparates the two halves of the stem, potentially eliminating thestructure via unfavorable loop entropy and allowing efficient firststrand synthesis.

The CD7 gene is not rearranged

Southern blot analysis of genomic DNA from placenta, peripheral bloodlymphocytes, T cells, HeLa cells, or the tumor lines used in the RNAblot analysis above showed identical Dra 1 digest patterns. Thus, theCD7 gene is not grossly altered during development, and the high levelof expression in the PEER cell line is not the consequence of asubstantial genomic rearrangement.

COS cells expressing CD7 do not bind IgM

Human peripheral blood T lymphocytes express receptors for IgMantibodies (FcRu: Moretta et al., Eur. J. Immunol. 5:565-569 (1975);McConnell et al., Immunol. 30:835-837 (1976)). Recently it has beenreported that CD7 might play a role in IgM binding by T cells (Sandrinet al., Leukocyte Typing III, Oxford Univ. Press, publisher, Oxford,England (1987)). L cells, normally CD7⁻ and FcRu⁻, become CD7⁺ and FcRu⁺when transfected with a 16 kb genomic fragment encoding the CD7 antigen(Sandrin et al., Leukocyte Typing III, Oxford Univ. Press, publisher,Oxford, England (1987)). Furthermore, IgM binding to CD7-positive cellscan be blocked by the anti-CD7 monoclonal antibody Huly-m2 (Thurlow etal., Transplantation 38:143-147 (1984)), and IgM columns bind a 37 kdprotein from radiolabeled lysates of peripheral blood T lymphocytes(Sandrin et al., Leukocyte Typing III, Oxford Univ. Press, publisher,Oxford, England (1987)).

Accordingly, COS cells expressing CD7 were tested for their ability tobind IgM. IgM receptor activity was assayed either by direct binding(Hardin et al., Proc. Natl. Acad. Sci. USA 76:912-914 (1979)) or by arosette assay with ox erythrocytes coated with an IgM fraction of rabbitanti-bovine red cell serum as described by Ercolani et al., J. Immunol.127:2044-2051 (1981)). Cells expressing CD7 neither bound human IgM norformed rosettes with IgM-coated erythrocytes. Under the same conditions,COS cells transfected with a cDNA encoding the human IgG receptor CDw32bound IgG directly and formed rosettes with IgG-coated erythrocytes.Erythrocytes coated with IgM or IgG antibodies also adhered to afraction of peripheral blood lymphocytes as reported (Moretta et al.,Eur. J. Immunol. 5:565-569 (1975)).

These results do not support the notion that the CD7 antigen is byitself an IgM receptor, although they do not exclude the possibilitythat COS cells suppress IgM binding activity in some manner, or that CD7is part of, or modified to become, an IgM receptor. That CD7 is not byitself an IgM receptor is supported by the observation that a number ofCD7⁺ T cell lines are FcRu-(Sandrin et al., Leukocyte Typing III, OxfordUniv. Press, publisher, Oxford, England (1987)).

EXAMPLE V Isolation and Molecular Cloning of the Human CDw32 Antigen

A cDNA encoding the human CDw32 antigen, a human receptor forimmunoglobulin G constant domains (Fc receptor), was isolated by themethod of the present invention, by virtue of its affinity for itsligand, IgG. The sequence of the isolated clone is most closely relatedto the murine beta 2 Fc receptor, but has diverged completely in theportion encoding the cytoplasmic domain. The receptor expressed in COScells shows a preference for IgG₁ among IgG subtypes, and no affinityfor IgM, IgA or IgE.

To isolate the Fc receptor clone, cDNA libraries were prepared fromtumor cell lines or from a human tumor and transfected into COS cells.After 48 hours, the cells were treated with mouse or human IgGantibodies, and allowed to settle on dishes coated withaffinity-purified sheep anti-mouse IgG or goat anti-human IgGantibodies. After lysis, DNA recovery, and transformation in E. coli,the cycle was repeated for two more rounds. Although no positive cloneswere isolated from the tumor line libraries, a cDNA clone encoding an Fcreceptor was isolated from a library prepared from a human adrenaltumor. It has been discovered that many tumors are heavily infiltratedby macrophages and lymphocytes. Thus, tumor RNA may be a productivesource in general for transcripts of human macrophages.

By indirect immunofluorescence assay, the human receptor expressed onCOS cells bound all mouse and human IgGs with relatively low affinity-10⁻⁷ M), and a clear discrimination was noted among human antibodiesfor IgG₁. Human IgM, IgA₁, IgA₂, and IgE did not bind, nor did murineIgM or IgA. As expected, human Fc, but not Fab fragments, bound to thetransfected cells. Among monoclonal antibodies donated to the ThirdInternational Workshop on Leukocyte Differentiation Antigens, three gavestrong positive immunofluorescence: two (out of two) recognizing the FcReceptor CDw32 determinant, and one (out of four) recognizing the CD23(B cell IgE Fc receptor) determinant. Monoclonals recognizing the Tcell/Macrophage Fc receptor antigen CD16 gave only weakimmunofluorescence comparable to that shown by control ascites.

Radioimmunoprecipitation of transfected COS cells with CDw32 antibodiesshowed the presence of a single 40 kd species, comparable in size to theantigen recognized on the surface of the myeloid CDw32⁺ line HL-60, andto the less abundant antigen present on the histiocytic leukemia lineU937. This result reinforces the notion that the isolated receptor isCDw32, as the CD16 receptor is reported to be substantially larger(60-70 kd).

The nucleotide sequence of the isolated receptor (FIGS. 9A-9B) is highlyhomologous to that of members of the recently isolated murine receptorfamily, and most closely related to the murine beta₂ receptor by nucleicacid homology. Surprisingly, the murine beta₂ receptor is found on T andB lymphocytes and macrophages, while the alpha receptor is restricted tomacrophages; in the human system, CDw32 (shown here to be beta₂ -like)is restricted to macrophages while another Fc receptor (CD16) is foundon lymphocytes and macrophages. The human sequence appears to havediverged from the mouse sequence by insertion of approximately 1 kb ofDNA a few bases 3' to the junction between the transmembrane andcytoplasmic domains. The junctions of the insertion site do not showobvious relationships to splice donor and acceptor sequences. Comparisonof the human and murine peptide sequences showed that the peptidesequence diverges at the end of the transmembrane domain, before thenucleotide sequence diverges, suggesting the existence of a selectivepressure favoring the creation of a differenct cytoplasmic domain.

RNA blot analysis showed that myeloid but not lymphocytic cell linesexpressed RNA homologous to the CDw32 probe. DNA blot analysis showedmultiple bands consistent with the existence of a small multigenefamily.

EXAMPLE VI Isolation and Molecular Cloning of Two cDNA Clones Encodingthe B Lymphocyte-specific CD20 (B1, Bp35) Antigen

Recent studies suggest that the pan B cell antigen CD20 (B1, Bp35) playsan important role in B cell activation. Monoclonal antibodies (mAb) toCD20 induce different cellular responses depending on the antibody usedand the stage of differentiation or activation of the target B cells.The monoclonal antibody 1F5 activates resting B cells by initiating thetransition from the G₀ to the G₁ phase of the cell cycle, and inducesdense tonsillar B cells to proliferate (Clark et al., Proc. Natl. Acad.Sci USA 82:1766 (1985); Clark and Shu, J. Immunol. 138:720 (1987)).However, 1F5 does not induce an increase in cytoplasmic free calcium anddoes not induce circulating B cells to proliferate (Rabinovitch et al,In: Leukocyte Typing III (McMichael, Ed.), p. 435, Oxford UniversityPress (1987)). Other anti-CD20 mAbs, such as B1, have been shown toblock B cell activation (Tedder et al., J. Immunol. 135:973 (1985)) andboth 1F5 and B1 can inhibit B cell differentiation (Golay et al., J.Immunol. 135:3795 (1985)). Recently it has been suggested thatphosphorylation and internalization of CD20 may be necessary steps for Bcell entry into the G₁ phase of the cell cycle (Valentine et al., In:Leukocyte Typing III (McMichael, Ed.), p. 440, Oxford University Press(1987)). In the present example, two CD20 cDNA clones were isolated andexpressed using the methods of the present invention.

Preparation of cDNA Library and Recovery of cDNA Clones by Panning

Poly(A)⁺ RNA was prepared from the human Burkitt cell line Daudi byoligo (dT) cellulose chromatography of total RNA isolated by proceduresdescribed herein. cDNA preparation and expression library constructionwere carried out as described.

Anti CD20 mAbs 1F5, 2H7, B1, L27, G28-2, 93-1B3, B-C1, and NU-B2 wereobtained from the International Leukocyte Typing Workshop (Valentine etal., In: Leukocyte Typing III (McMichael, Ed.), p. 440, OxfordUniversity Press (1987)). Purified mAbs were used at a concentration of1 ug/ml and ascites were used at a dilution of 1:1000. Panning was doneaccording to the present method. In the first round of screening, eight10 cm dishes of 50% confluent COS cells were transfected by theDEAE-Dextran method. Subsequent screening cycles were performed byspheroplast fusion.

Immunoprecipitation, Sequencing, RNA and DNA Blot Hybridization

B cell lines CESS and Daudi were metabolically labeled with ³⁵S-methionine and ³⁵ S-cysteine for 6 h at 37° C. COS cells transfectedby the DEAE-Dextran method were similarly labeled 36 hourspost-transfection. The labeled cells were incubated with the B1 mAb(Coulter) at 4° C. for 1 h, washed in PBS, and lysed with 0.5% NP-40,0.1% SDS, 0.05% deoxycholate and 1 mM PMSF in PBS. After centrifuging(13000×g, 5 min.), the lysate was incubated with fixed S. aureus cells(Calbiochem) for 1 hr at 4° C. The S. aureus cells were pelleted, washed5 times with 1% NP-40/PBS, eluted and electrophoresed through 12.5%polyacrylamide gels.

DNA and RNA blot analysis and hybridization probe preparation werecarried out as described. Sequencing was done by the method of Sanger etal., Proc. Natl. Acad. Sci. USA 74:5463 (1977)). The nucleotide sequenceof the CD20.4 cDNA is represented in FIGS. 10A-10B.

Two cDNA clones, bearing inserts of 1.5 (CD20.4) and 1.0 kb (CD20.6),were isolated from a Daudi cell DNA library by panning with a panel ofmAbs against CD20. COS cells transfected with either clone reacted withall members of the panel of antibodies. Immunoprecipitation of thecDNA-encloded protein from transfected COS cells showed two bands of 32and 30 kd reminiscent of the 37 and 35 kd bands observed in different Bcell subsets and lines (Valentine et al., "Structure and Function of theB Cell Specific 35-37 kDa CD20 Protein," In: Leukocyte Typing III, A.McMichael et al., eds., Oxford University Press, p. 440 (1987)). It hasbeen the experience of the present inventors that the molecular massesof surface antigens expressed in COS cells are consistently smaller thanthose of their native counterparts. This may be due to differences inglycosylation.

Both cDNA clones have the same coding sequence, and differ only in the3' untranslated region. The insert in clone CD20.6 has a short polyAtail and lacks a consensus polyadenylation signal, while the insert inCD20.4 lacks a polyA tail and extends 431 bp beyond the 3' terminus inCD20.6 (FIGS. 10A-10B).

RNA blot analysis showed that three transcripts of 3.8, 3.0 and 1.5 kbwere present in B cells but absent from other cell types, in agreementwith the known pattern of antibody reactivity (Clark et al., Proc. Natl.Acad. Sci. USA 82:1766 (1985); Clark et al, J. Immunol. 138:720 (1987);Tedder et al., J. Immunol. 135:973 (1985); Golay et al., J. Immunol.135:3795 (1985)). It appears likely that the CD20.6 clone is derivedfrom the 1.5 kb transcript or possibly from an even shorter,undetectable species. Because the CD20.4 clone lacks a poly(A)⁺ tail,its source cannot be inferred at present.

DNA blot analysis showed that the CD20 genomic sequences are notrearranged during development and are not amplified in the cell linesexamined. A restriction fragment length polymorphism was observed in aDNA sample obtained from placenta.

The amino acid sequence predicted by the cDNA contains 297 residues andhas a molecular mass of 33,097 daltons. The sequence contains threemajor hydrophobic stretches involving residues 51-103, 117-141 and183-203 (FIG. 10C). Two other notable characteristics are the absence ofan amino-terminal signal peptide and the presence of a highly chargedcarboxy-terminal domain. A polyclonal anti-CD20 antibody that recognizedthe last 18 residues of the carboxy-terminus reacts with lysates ofcells expressing CD20 but not with intact cells, suggesting that theCD20 carboxy terminus is located within the cytoplasm. Since there is noamino-terminal signal peptide, it is likely that the amino-terminus isalso intracellular, and that the first hydrophobic region acts as aninternal membrane insertion signal (Zerial et al., EMBO J. 5:1543(1986)). The first hydrophobic region is composed of 53 residues and istherefore long enough to span the membrane twice if organized as analpha helix. Because there are two remaining hydrophobic regions, theintracellular localization of the carboxy-terminus requires that thefirst hydrophobic domain exit the membrane on the side. Alternatively,the carboxy-terminal antibody may only recognize epitopes exposed bydetergent treatment allowing the carboxy-terminus to be extracellularand forcing the first hydrophobic domain to exit the membrane on theextracellular side. The sequence contains 2 potential N-glycosylationsites (Asn-Xaa-Ser/Thr, where Xaa cannot be Pro (Bause, Biochem. J.209:331 (1983)) at positions 9 and 293, but neither of these is expectedto be used if located in intracellular domains of the molecule. Thedifference in molecular mass between CD20 expressed on COS cells and onB cells is therefore presumably due to O-linked glycosylation, althoughother forms of post-translational modification are not excluded. If thecarboxy-terminus is intracellular, the only extracellular domain wouldlie between residues 142 and 182. This region is rich in serine andthreonine residues which might support O-glycosylation.

The observation of two protein species in COS cells cannot be explainedby alternate splice formation because the cDNA sequence does not containany promising splice donor or acceptor sequences (Shapiro et al., Nucl.Acids Res. 15:7155 (1987)). A difference in glycosylation or alternatetranslational initiation site selection may account for the two speciesobserved. Initiation at either the first or the second ATG gives proteinmolecular masses of 33.1 and 30.8 kd respectively, in good agreementwith the sizes observed in COS cells. Neither ATG is embedded in theconsensus sequence proposed by Kozak (Nucl. Acids Res. 12:857 (1984)).Use of alternate initiation sites has been reported for several proteins(Kozak, Nucl. Acids Res. 12:857 (1984)).

Comparison of the peptide sequence with the sequences in the NationalBiomedical Research Foundation database showed no significant homologyby the FASTP rapid sequence alignment algorithm. Because the bulk of theprotein appears to be confined to the interior of the membrane and thecell, it seems plausible that it may play a role in transducing signalsfrom other transmembrane proteins to the cell interior. Consistent withthis role is the relatively hydrophilic nature of the hydrophobicregions which might allow hydrogen bond interactions with thetransmembrane portions of other proteins.

EXAMPLE VII Isolation and Molecular Cloning of ICAM, An Adhesion Ligandof LFA-1

Antigen-specific cell contacts in the immune system are strengthened byantigen-non-specific interactions mediated in part by lymphocytefunction associated or LFA antigens (Springer, T. A., et al., Annu. Rev.Immunol. 5:223-252 (1987); Anderson, D. C., et al., Annu. Rev. Medicine5:175-194 (1987)). The LFA-1 antigen, a major receptor of T cells, Bcells and granulocytes (Rothlein, R., et al., Exp. Med. 163:1132-1149(1987)), is involved in cytolytic conjugate formation,antibody-dependent killing by NK cells and granulogytes, and helper Tcell interactions. LFA-1 has been placed in the integrin family of cellsurface receptors by virtue of the high sequence similarity between theLFA-1 and integrin beta chains (Kishimoto, T. K., et al., Cell48:681-690 (1987); Hynes, R. O. Cell 48:549-554 (1987)). The adhesionligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp(RGD) sequence motif, e.g., fibronectin, fibrinogen, vitronectin and vonWillebrand factor (Ruoslahti, E., et al., Cell 44:517-518 (1987)).

In this example, the Intercellular Adhesion Molecule-1 (ICAM-1), aligand for LFA-1 (Rothlein, R., et al., J. Immunol. 137:1270-1275(1986); Dustin, M. L., et al., J. Immunol. 137:245-254 (1986)), wascloned according to the methods of the present invention. ICAM containsno RGD motifs, and instead is homologous to the neural cell adhesionmolecule NCAM (Cunningham, B. A., et al. Science 236:799-806 (1987);Barthels, D., et al., EMBO J. 6:907-914 (1987)). COS cells transfectedwith the ICAM cDNA clone bind myeloid cells by a specific interactionwhich can be blocked by monoclonal antibodies directed against eitherLFA-1 or ICAM-1.

A cDNA library was constructed using RNA prepared from HL60 cellsinduced with phorbol myristyl acetate (PMA). The library was transfectedinto COS cells and cells expressing surface antigens were recoveredaccording to the methods of the present invention by panning with theanti-ICAM monoclonal antibodies (mAbs) 8F5 and 84H10 (McMichael, A. J.,et al., eds., Leukocyte Typing III. White Cell Differentiation Antigens,Oxford University Press (1987)). Episomal DNA was recovered from thepanned cells and the expression-panning cycle repeated a further 2 timesto obtain a cDNA clone designated pICAM-1.

COS cells transfected with pICAM-1 gave positive surfaceimmunofluorescence reactions with three anti-ICAM-1 antibodies: 8F5;84H10; and RR-1. Immunoprecipitation of pICAM-1-transfected COS cellswith the mAb 84H10 gave a band of molecular mass 100 kd. 30). A slightlylarger protein of 110 kd was precipitated from HL60 cells induced for 48hours with either phorbol myristyl acetate (PMA), gamma-interferon(gammaIFN), tumour necrosis factor (TNF), or interleukin-1 beta (IL-1beta), but was absent from uninduced cells. The smaller molecular massof ICAM-1 expressed in COS cells is consistent with the lower molecularmasses observed for other surface antigens expressed in COS cells.

RNA blot analysis showed 2 species of 3.2 kb and 1.9 kb present in HL60cells stimulated with either PMA, gamma IFN, TNF or IL-1 gamma, butabsent in uninduced cells. Thus, the expression of ICAM-1 is regulatedby a number of cytokines, apparently at the level of transcription.Similar species were present in B cells (JY and Raji), T cells (Peer andT blasts) and Lymphokine Activated Killer cells (LAK). The structure ofthese ICAM-1 transcripts and their relationship to the pICAM-1 cDNAremains to be established. Blot hybridization of genomic DNA fromplacenta revealed a pattern consistent with a single copy gene.

To investigate whether pICAM-1 encodes a functional cell adhesionmolecule, COS cells expressing ICAM-1 were tested for their ability tobind HL60 cells. After 30 minutes at 37° C. in the presence of Mg²⁺,HL60 cells strongly adhered to the ICAM-expressing COS cells, but not tomock transfected cells. The specificity of this adhesion wasdemonstrated by preincubating the ICAM-1 expressing COS cells with mAb84H10. All HL60 binding was abolished under these conditions. An isotypematched monoclonal antibody, W6/32, which recognizes a monomorphicHLA-ABC related determinant of approximately equal abundance to ICAM-1on transfected COS cells, had no effect on the adhesion. Similarly,preincubation of the HL60 cells with either 84H10 or W6/32 did notinhibit binding.

To determine if LFA-1 was acting as the receptor for ICAM-1 in thissystem, HL60 cells were pretreated with antibodies against the betachain of LFA-1 (CD18 (McMichael, A. J., et al., eds., Leukocyte TypingIII. White Cell Differentiation Antigens, Oxford University Press(1987))) and then subjected to the binding assay. All adhesion toICAM-expressing COS cells was blocked. Pretreatment of COS cells withthe CD18 antibodies had no effect on the adhesion. This provides directevidence that ICAM-1 is indeed acting as an adhesion ligand for LFA-1.

The sequence of the pICAM-1 cDNA insert consists of 1846 nucleotides(FIGS. 11A-11C). The predicted peptide sequence of 532 residues has thetypical features of a transmembrane protein including a putative signalsequence, which may be cleaved between glycine-25 and asparagine-26 (vonHeijne, G., Nucl. Acids Res. 14:4683-4690 (1986)), and a single 25residue membrane-spanning domain terminating in a short, highly chargedcytoplasmic domain. The extracellular domain contains seven potentialN-linked glycosylation sites which could adequately explain thedifference in size between the deglycosylated precursor (55 kd) and thefinal product (90-115 kd) (Dustin, M. L., et al., J. Immunol.137:245-254 (1986)). Differential use of these putative glycosylationsites could also explain the heterogeneous molecular mass of ICAM-1observed in different cell types (Dustin, M. L., et al., J. Immunol.137:245-254 (1986)).

LFA-1 is a member of the integrin family of cell surface receptors(Kishimoto, T. K., et al., Cell 48:681-690 (1987); Hynes, R. O., Cell48:549-554 (1987)). The tripeptide motif Arg-Gly-Asp (RGD) is a commonfeature of the ligands for this family, e.g., fibronectin, fibrinogen,vitronectin and von Willebrand factor, and is crucial forligand-receptor interaction (Ruoslahti, E, et al., Cell 44:517-518(1987)). However, ICAM-1 contains no RGD motifs, bearing instead asingle RGE sequence at position 152. A search of the National BiomedicalResearch Foundation (Dayhoff, M. O., et al., Methods Enzymol. 91:524-545(1983)) (NBRF) database revealed no significant similarities to otherproteins. However, a comparison to a laboratory database containingrecently published surface proteins did reveal a surprising andsignificant similarity between ICAM-1 and the neural cell adhesionmolecule NCAM-1 (Cunningham, B. A., et al., Science 236:799-806 (1987);Barthels, D., et al., EMBO J. 6:907-914 (1987)). The optimal alignmentscore obtained using the NBRF ALIGN program is 8 standard deviationsabove the mean score obtained from 500 random permutations of thesequences. The probability of the spontaneous occurrence of an equal orhigher score is approximately 10⁻⁹.

Using a database of known immunoglobulin related sequences, it has beenshown that ICAM-1 may be divided into five Ig domains (28-112, 115-206,217-310, 312-391, and 399-477) each of which shows significantsimilarity with other members of the Ig superfamily (Williams, A. F.,Immunol. Today 8:298-3-3 (1987)). For example, domain I is similar toCD3 while domains IV and V are similar to domains of myelin associatedglycoprotein (Arguint, M., et al., Proc. Natl. Acad. Sci. USA 84:600-604(1987)) and carcinoembryonic antigen (Beauchemin, N., et al., Mol. Cell.Biol. 7:3221-3230 (1987)). All five Ig domains of NCAM align with the Igsegments in ICAM, and the principal contribution to the similarity comesfrom domains II and III of ICAM. Finally, the T cell-specific adhesionmolecule CD2 shows roughly the same similarity to NCAM as does ICAM, butICAM and CD2 are only weakly related. Thus, some precursor of NCAM isancestral to both ICAM and CD2.

Through its cell adhesion to LFA-1, ICAM can mediate migration oflymphocytes into areas of inflammation. Inhibiting such migration byblocking ICAM binding to LFA-1 could reduce or inhibit inflammation.Such inhibition could be affected by small organic molecules, i.e.,drugs, identified in an ICAM streaming assay. Fusion proteins composedof the extracellular domain of ICAM and IgG molecules are suitable foridentifying such inhibitors. Likewise, compounds that interfere withICAM binding to Rhinovirus or Plasmodium falciparium can be identifiedby analogous methods.

The Applicants have constructed fusion proteins consisting of the Igdomains of the ICAM-1 extracellular domain, which are useful instreaming assays. The CH2 and CH3 domains of IgG1 were fused toextracellular Ig domain 1, domains 1 and 2, domains 1-3, domains 1-4,and domains 1-5 of ICAM-1. The corresponding clones have been designatedICAM1-Eγ1, ICAM1-Eγ2, ICAM1-Eγ3, ICAM1-Eγ4 and ICAM1-Eγ5. The first twoN-terminal domains (amino acids 28-112 and 115-206, respectively) arebelieved to be most useful in a binding assay to identify compounds thatinhibit ICAM binding to another ligand.

Soluble ICAM can also be used directly to interfere with the binding ofcellular ICAM to Rhinovirus or Plasmodium, thereby inhibiting theinfection. Soluble ICAM includes the extracellular domain of ICAM, orfunctional derivatives thereof, in truncated form or fused to a solubleprotein such as Ig.

For the purpose of the invention as it relates to ICAM-1 protein, theterm "functional derivatives" includes polypeptides that have at leastabout 80% amino acid identity to the entire disclosed ICAM-1 amino acidsequence, its intracellular domain, its extracellular domain (aminoacids 1-477) or any of immunoglobulin domains 1-5 therein, or thesequence comprising the LFA-1 binding site, and that have a bindingaffinity to a ligand of ICAM-1 such as LFA-1 that is at least about 30%as that of the disclosed sequence. Such polypeptides may optionally beincluded as part of a larger protein. Increasingly preferred are aminoacid identities that increase integrally, i.e., at least about 81%, 82%,83%, etc. Binding affinity of ICAM-1 to a ligand such as LFA-1 can bedetermined by methods known in the art. Increasingly preferred arebinding affinities that increase in increments of 10%, i.e., at leastabout 40%, 50%, etc., of that of the disclosed sequence.

A nucleotide sequence is a "functional derivative" of ICAM-1 if itencodes the diclosed ICAM-1 amino acid sequence or a functionalderivative thereof.

In designing functional derivatives of ICAM-1 protein, amino acids orregions known to be important to the binding of ICAM-1 to a ligand suchas LFA-1 should be highly conserved. Publications relating to binding ofICAM-1 to LFA-1 include: Hedman, H. & Ludman, E. (1991) J. Immunol.149:2295-9; Dang, L & Rock, K. (1991) J. Immunol. 146:3273-9; Ross, L etal. (1992) J. Biol.Chem. 267:8537-43; Hibbs, M. et al. (1991) Science251:1611-3; Berendt, A. et al. (1992) Cell 68:71-81; Ockenhouse, C. etal. (1992) Cell 68:63-9; Diamond, M. et al. (1991) Cell 65:961-71;Cabanas, C. & Hogg, N. (1991) FEBS Lett 292:284-8; and Staunton, D. etal. (1990) Cell 61:243-54 (errata at Cell (1990) 61:1157 and Cell (1991)667:1312).

Additional publications relating to function of ICAM-1 include: Salkind,A. et al. (1991) J. Clin. Invest. 88:505-11; Fischer, H. et al. (1992)J. Immunol. 148:1993-8; Carlow, D. et al. (1992) J. Immunol.148:1595-606; Rothlein, R. et al. (1991) J. Immunol. 147:3788; Fine, J.& Kruisbeek, A. (1991) J. Immunol. 147:2852-9; Tang, A. & Udey M. (1991)J. Immunol. 146:3347-55; Symington, F. & Santos, E. (1991) J. Immunol.146:2169-75; Rosenstein, Y. et al. (1991) Nature 354:233-5; Fujiota, H.et al. (1991) Biochem. Biophys. Res. Commun. 177:664-72; Nambu, M. etal. (1992) Cell Immunol. 143:335-47; Sanders, V. & Vitetta, E. (1991)Cell Immunol. 132:45-55; Diamond, M. et al. (1990) J. Cell Biol.111:3129-39; Maraskovsky, E. et al. (1992) Int. Immunol. 4:475-85;Padros, M. et al. (1992) Clin. Exp. Immunol. 88:329-34; and VanSeventer, G. et al. (1991) Eur. J. Immunol. 21:1711-18.

In particular, Piela-Smith, T. et al. (1992) J. Immunol. 148:1375-81;Staunton, D. et al. (1990) Cell 61:243-54 (errata at Cell (1990) 61:1157and Cell (1991) 667:1312); Staunton, D. et al. (1989) Cell 56:849-53;Greve, J. et al. (1989) Cell 56:839-47; Staunton, D. et al. (1992) J.Immunol. 148:3271-4; Register, R. et al. (1991) J. Virol. 65:6589-96;Greve, J. et al. (1991) J. Virol. 65:6015-23; and Lineberger, D. et al.(1992) Virus Res. 24:173-186, discuss binding of ICAM-1 to rhinovirus.Oppenheimer-Marks, N. et al. (1991) J. Immunol. 147:2913-21; and Perry,M. et al. (1992) Cell Tissue Res. 268:317-26; describe the role ofICAM-1 in transendothelial migration, and Dustin, M. et al. (1992) J.Immunol. 148:2654-63, describe B cell migration on ICAM-1 coatedsubstrates. Webb, D. et al. (1991) J. Immunol. 146:3682-6, describe therole of ICAM-1 in monocyte invasion of tumors. Berendt, A. et al. (1992)Cell 68:71-81; Ockenhouse, C. et al. (1992) Cell 68:63-9; andOckenhouse, C. et al. (1991) J. Infect. Dis. 164:163-169, discuss ICAM-1in its role as a receptor for Plasmodium falciparum. Gruber, M. et al.(1991) AIDS Res. Hum. Retroviruses 7:45-53, discuss the role of ICAM-1in HIV syncytia formation. Dohlsten, M. et al. (1991) Eur. J. Immunol.21:131-5, discuss the role of ICAM-1 in staphylococcalenterotoxin-mediated cytotoxicity.

EXAMPLE VIII Isolation and Molecular Cloning of the Human CD19, CD20,CDw32a, CDw32b and CD40 Antigens

The rapid immunoselection cloning method of the present invention wasapplied to isolate and clone the CD19, CD20, CDw32a, CDw32b, and CD40antigens. The nucleotide sequence of CD19is shown in FIGS. 12A-12B. Thenucleotide sequence of CD20 is shown in FIGS. 13A-13B. The nucleotidesequence of CDw32a is shown in FIGS. 14A-14B. The nucleotide sequence ofCDw32b is shown in FIGS. 15A-15B. The nucleotide sequence of CD40 isshown in FIG. 16.

EXAMPLE IX Cloning, Sequence and Expression of CD36

To isolate a cDNA clone encoding CD36, a human placenta cDNA (Simmonsand Seed (1988) Nature 333:568-570) was transferred into COS cells usingDEAE-Dextran as a facilitator (Example I, supra). 48 hoursposttransfection the cells were detached from the dishes withouttrypsin, incubated with monoclonal anti-CD36antibodies 5F1 (Bernstein etal. (1982) J. Immunol. 128:876-881) (Andrews et al. (1984) J. Immunol.128:398-404) and panned on dishes coated with goat anti-mouseimmunoglobulin antibodies. Nonadherent cells were removed by gentlewashing, the adherent cells were lysed, and episomal plasmids recoveredfrom the cells were purified and transformed into E. coli. After twosimilar rounds of enrichment following spheroplast fusion, plasmid DNAsrecovered from 11 out of 12 randomly chosen colonies were found todirect the appearance of CD36 determinants in transfected COS cells.

Two of the clones were chosen at random for further analysis. Both boreinserts of about 1.9 kb, and showed identical restriction enzymefragment patterns. COS cells transfected with either of these clonesreacted with monoclonal antibodies 5F1 and F13, and with OKM5 (Ortho,Raritan, N.J.). Immunoprecipitation of transfected cells with a pool ofanti-CD36 antibodies revealed the presence of an 83 kd molecule presenton transfected COS cells and C32 melanoma cells, and absent from control(CD25-transfected) COS cells. A high molecular weight species, possiblydimeric CD36, was immunoprecipitated from the transfected COS celllysate, but not from the C32 cell lysate. The nucleotide sequence isgiven in Table 1. In Table 1, the nucleotide sequence numbering is shownin the left margin at the beginning of each line. The deduced amino acidsequence is shown as single letter code underneath the beginning of eachcoding nucleotide triplet, with the initiation methionine indicated bythe number 1 above the initiator codon. The potential sites of N-linkedglycosylaton in the derived amino acid sequence are underlined by asingle dashed line. The putative transmembrane domain isdouble-underlined. Although two consensus polyadenylation (AATAAA)motifs are found in the cDNA, there is no poly(A) tail, and none of theRNA species observed by blot hybridization are short enough tocorrespond to polyadenylation at these sites, assuming that thetranscripts bear the same approximate 5' end as observed in the clone.The presumed initiation codon is not the first ATG found in the clone,but the previous two are closely followed by in-frame terminationcodons. The predicted initiator methionine is followed by a shorthydrophobic region resembling a secretory signal sequence for which,however, no clear identification of the cleavage site can be made. Therecent determination of Table 1 the amino terminal 36 amino acidsequence (Tandon et al. J. Biol. Chem. 264:7570-7575 (1989)) indicatesthat the mature polypeptide begins at the amino acid residue immediatelyfollowing the initiator methionine. It is not clear whether the singleArg residue preceding the hydrophobic region would be sufficient toallow amino terminal membrane anchoring. The resulting polypeptidepossesses 471 residues with a predicted molecular weight of about 53 kd.The proposed extracellular domain is followed by 27 predominantlyhydrophobic residues corresponding to a transmembrane domain, and 6residues (of which 3 are basic) corresponding to an attenuatedintracellular domain. The presence of 10 potential N-linkedglycosylation sites appears sufficient to account for the discrepancy inmolecular mass between the predicted polypeptide and the 83 kd speciesfound by immunoprecipitation. No significant homology was detectedfollowing comparison of the entire sequence to various databases ofknown proteins. However some internal structure was apparent. All of thecysteine residues in the extracellular domain are confined to a domaindefined by residues 937 to 1209 of the nucleotide sequence. Taking thecysteine placement as a guide, the extracellular domain could be dividedinto three segments, in which two domains without cysteine preceded andfollowed the cysteine rich segment. However sequence comparisons withthese segments did not show any significant relatedness to othermolecules in existing databases, nor, in particular, to thrombospondin.

The CD36 protein was purified by immunoprecipitation. Since the rapidimmunoselection cloning method depended upon expression of transfectedCOS cells, it follows that the same cell lines from which CD36 cDNA wascloned could also be used as a source of the expressed protein.

    TABLE 1                                                                          -                                                                              ##STR1##                                                                       ##STR2##                                                                       ##STR3##                                                                       ##STR4##                                                                       ##STR5##                                                                       ##STR6##                                                                       ##STR7##                                                                       ##STR8##                                                                       ##STR9##                                                                       ##STR10##                                                                      ##STR11##                                                                      ##STR12##                                                                      ##STR13##                                                                      ##STR14##                                                                      ##STR15##                                                                      ##STR16##                                                                

C32 melanoma cells, CD36 transfected cells COS cells, and CD25 (control)transfected COS cells were surface labelled with Na¹²⁵ I and lysed in aphosphate buffered saline solution containing 1 mM phenylmethylsulfonylfluoride, 0.5% NP-40 and 0.1% sodium dodecyl sulfate. Anti-CD36monoclonal antibodies were added, and allowed to absorb to the lysatefor 12 hours at 4° C., after which goat anti-mouse Immunoglobulin beads(Cappel) were added, mixed for two hours, and washed as described (Clarkand Einfeld J. Immunol. 135:155-167 (1986)). Larger amounts of proteincan also be obtained in purified form from a transfected COS cell lysateby an immunoaffinity column purification. Other antibodies to CD36 maybe obtained, using CD36 protein, expressed and/or purified as described,as immunogen.

CD36 has been identified as a binding site for cytoadherence ofPlasmodum falciparum parasitized erythrocytes, by the inventors hereinand by Ockenhouse, C. D. et al. (1989) Science 243:1469-1741.Cytoadhesion of parasitized erythrocytes has been shown to be blocked bymonoclonal antibodies to CD36. Incubation of infected erythrocytes withCOS cells transfected with a CD36 cDNA showed pronounced cytoadherence.The ability of P. falciparum parasitized erythrocytes to evade splenicclearance by adherence to peripheral vascular beds is thought to play animportant role in the pathogenicity of Falciparum malaria and tocontribute to the lethal syndrome of cerebral malaria by causingocclusion of the small vessels of the brain. Therefore the cDNA andpurified protein of the present invention are useful for providingsufficient purified CD36 to make therapeutic monoclonal antibodies.

EXAMPLE X Isolation and Cloning of Three cDNA Clones EncodingMacrophage-Specific FcRI

Three independent cDNA clones (designated p135, p90 and p98/X2) encodinghuman FcRI were isolated by the rapid immunoselection cloning method ofthe present invention from a cDNA library expressed in COS cells. (Seealso Allen, J. M. and Seed B., Science 243:378-381 (1989)). The cDNAlibrary was constructed from polyadenylated RNA obtained from cells of asingle patient undergoing extracorporeal interleukin-2 inductiontherapy. Expression of the three cDNAs in COS cells gave rise to IgGbinding of the appropriate affinity and subtype specificity. DNAsequence analysis revealed that the cDNAs encode similar type I integralmembrane proteins with 3 extracellular immunoglobulin domains. Theintracellular domain of p98/X2 diverges from that of the other twocDNAs. A composite sequence of the three cDNAs is shown in Table 2 withthe nucleotide differences of the p89/X2 or p90 clones shownrespectively below or above the p135 sequence. Dashes denote gaps and noresidues are shown above or below where the sequences are identical. Thep90 cDNA has the shortest 5' untranslated region, 7 additional residuesbetween the polyadenylation motif and the poly A tract, and 2polymorphisms in the coding region. The p98/X2 cDNA has the longest 5'untranslated region, 1 polymorphism in the coding sequence, and divergesfrom the other two cDNAs at residue 1051, becoming a complex pattern ofrepeats of upstream sequences. The p98/X2 clone lacks a polyadenylationsite.

The FcRI protein from each of the three clones was purified from therespective COS cell lines which expressed them, by immuno-adsorption toIgG-agarose. (See Stengelin S. et al., EMBO J. 7:1053 (1988)). Gelelectrophoresis of purified proteins showed a single species from p135and p90 COS cells, relative molecular size 70 kd. Cells transfected withp98/X2 expressed a protein of 67 kd. A slightly larger protein of 75 kdwas adsorbed from untreated and interferon-gamma-treated U937promonocyte cells. The smaller mass observed in COS cells is consistentwith the reduced masses observed from Table 2 other surface antigensexpressed in COS cells, see e.g., Example IX.

The predicted polypeptide sequences show the typical features of a typeI integral membrane protein, and include a short hydrophobic signalsequence, a single 21-residue hydrophobic membrane-spanning domain, anda short, highly charged cytoplasmic domain (FIGS. 4A-4B). Theextracellular portion contains six potential N-linked glycosylationsites and six Cys residues distributed among three C2 set Ig-relateddomains.

FcRI is a high-affinity receptor for the Fc portion of IgG, normallylocated on the cell surfaces of macrophages. The ability to interferewith such bonding, or to cause it to occur on surfaces other thanmacrophages, is useful in therapy. For example, a fusion protein of FcRIand a receptor ligand will be helpful to increase the potency ofantibodies in therapy.

EXAMPLE XI Isolation and Cloning of cDNA Encoding T-Lymphocyte TLiSAAntigen

A cDNA clone encoding TLiSA1 was obtained from a human T-cell cDNAlibrary transferred into COS cell as described and subjected to therapid immunoselection cloning method of the invention. A monoclonalantibody ACT-T-SET TLiSA1 (T-Cell Sciences Corp., Cambridge, Mass.) wasused to detect transfected COS cells expressing the cloned cDNA, bypositive indirect immunofluorescence. The positive plasmid contained ina 1.7 kb insert.

    TABLE 2                                                                          -                                                                              ##STR17##                                                                     λ                                                                        ##STR18##                                                                      ##STR19##                                                                      ##STR20##                                                                     λ                                                                        ##STR21##                                                                      ##STR22##                                                                      ##STR23##                                                                      ##STR24##                                                                      ##STR25##                                                                      ##STR26##                                                                      ##STR27##                                                                     λλ                                                                ##STR28##                                                                

TLiSA protein was isolated by immunoprecipitation, as described supra,Example IX. The protein had a molecular weight of about 50 kd, asmeasured by gel electrophoresis.

The nucleotide sequence of the cDNA was determined by dideoxynucleotidechain termination as described, supra. The sequence of 1714 residues isgiven in Table 3, together with the deduced amino acid sequence shown insingle letter code under the first nucleotide of each coding triplet.The ATG encoding the presumed initiator methionine is followed by ashort hydrophobic region consistent with a secretory signal sequences,the most likely excision site being 19 residues into the open readingframe. The resulting polypeptide, if not further processed, wouldpossess 317 residues with a predicted molecular weight of about 36 kd.The proposed extracellular domain is followed by 25 predominantlyhydrophobic residues corresponding to the intracellular domain. (Table3, double underlined.) The presence of 9 potential N linkedglycosylation sites (Table 3, single dashed lines) appears sufficient toaccount for the discrepancy in molecular mass between the predictedpolypeptide and the 50 kd species found by immunoprecipitation.

TLiSA is involved in mediating IL-2 induced differentiation of T-cellsinto cytolytic forms. Antibodies to TLiSA are useful to prevent IL-2stimulated T cell differentiation, and to modulate adverse effects ofIL-2 in therapy.

EXAMPLE XII The Isolation and Molecular Cloning of cDNA Encoding for Blymphocyte-specific CD22 Antigen

To isolate a CD22 cDNA, an expression library was constructed from theBurkitt lymphoma cell line Daudi, introduced into COS cells by theDEAE-Dextran method described supra, and subjected to three rounds ofpanning and reintroduction

    TABLE 3                                                                          -                                                                              ##STR29##                                                                      ##STR30##                                                                      ##STR31##                                                                      ##STR32##                                                                      ##STR33##                                                                      ##STR34##                                                                      ##STR35##                                                                      ##STR36##                                                                      ##STR37##                                                                      ##STR38##                                                                      ##STR39##                                                                      ##STR40##                                                                      ##STR41##                                                                      ##STR42##                                                                      ##STR43##                                                                

into E. coli as described in Seed and Aruffo, Proc. Natl. Acad. Sci. USA84:3365-3369 (1987) and Aruffo and Seed, Proc Natl. Acad. Sci. USA84:8573-8577 (1987). Of 16 plasmids picked after the third round, twotested positive for CD22 expression by indirect immunofluorescence inCOS cells. Of the five carbohydrate-related epitopes, A, B, C, D and E,recognized by anti-CD22 monoclonal antibodies, only epitopes A and Dwere expressed in COS cells.

Immunoprecipitation of CD22 from transfected COS cells yielded a singleband corresponding to a molecular mass of 110 kd, smaller than the 135kd species obtained from Burkitt lymphoma Raji cells. The difference inmass may be related to differences in glycosylation. Since theimmunogenic epitopes of CD22 are carbohydrate-related, these differencesmight account for the absence of epitopes B, C and E.

RNA blot hybridization analysis has revealed the presence of a major 3kb RNA species and 4 minor species of 2.6, 2.3, 2.0 and 1.5 kb inseveral B cell lines. RNA encoding CD22 has not been found in several Tcell lines, including peripheral blood T cells, the T cell leukemiaJurkat, the myeloid leukemia lines HL60 and U937 and the hepatoma HepG2.

DNA blot hybridization of placental DNA gave a simple pattern consistentwith a single copy gene. DNA sequence analysis by the dideoxy methoddescribed supra showed that the 2107 bp insert encoded a polypeptide of647 amino acids. The nucleotide and amino acid sequences appear in Table4. The initial methionine is followed by 18 predominantly hydrophobicamino acids resembling a secretory signal sequence. The mature protein,having a relative molecular weight of 71.1 kd consists of anextracellular portion of 491 residues, followed by a 19 residuemembrane-spanning domain (doubly underlined), and an intracellulardomain of 118 amino acids. Ten potential N-linked glycosylation sites(N--X--S/T, X not equal to P) are found in the predicted extracellulardomain, as well as a large number of serine and threonine residues whichmay be sites of O-linked glycan addition. The abundance of potentialglycosylation sites and the difference in mass between the predictedprotein backbone and the product precipitated from COS cells and B celllines suggest that about 50% of the mass of CD22 is contributed bycarbohydrate.

                                      TABLE 4                                     __________________________________________________________________________     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                     ##STR56##                                                                     ##STR57##                                                                     ##STR58##                                                                     ##STR59##                                                                     ##STR60##                                                                     ##STR61##                                                                     ##STR62##                                                                     ##STR63##                                                                     ##STR64##                                                                     ##STR65##                                                                     ##STR66##                                                                     ##STR67##                                                                     ##STR68##                                                                    2001GCCTCAGGCACAAGAAAATGTGGACTATGTGATCCTCAAACATTGACACTGGATGGGCTGCAGCAGAGGC    C                                                                             2081GCGGGGGCAGGGAAGTCCCCGAGTTT                                                __________________________________________________________________________

The extracellular portion of CD22 consists of five segments havingIg-like domain organization. The short intercysteine spacing (63 and 64residues in domains 1 and 2, and 42 residues in domains 3-5) suggeststhat they fold into the 7 strand two layer beta-sheet structurecharacteristic of immunoglobulin constant regions rather than the 9strand structure of variable regions.

Because CD22 has been found to be highly homologous to myelin associatedglycoprotein (MAG), a neuronal cell surface protein which mediatescell-cell contacts during myelogenesis, it was postulated that CD22 hasa role in B cell adhesion. COS cells transfected with CD22 cDNA werecontacted with erythrocytes or peripheral blood mononuclear cells andincubated under conditions which minimized nonspecific interaction.Erythrocyte and mononuclear cell resetting was observed withCD22-positive COS cells but not with COS cells transfected with anunrelated cDNA clone.

B cell adhesion studies involving anti-epitope monoclonal antibodieshave indicated that different epitopes of CD22 may participate inerythrocyte and monocyte adhesion and that different ligands may berecognized on each cell type. B cell adhesion studies also suggest thatCD22, in a manner analogous to T cell CD2, CD4 and CD8 adhesion totarget cells, may promote recognition by the B cell antigen receptor byintensifying B cell-presenting cell contacts. CD22 has been previouslyimplicated in the transmission of signals synergizing with the antigenreceptor (Pezutto et al., J. Immunol. 138:98-103 (1987)) andcrosslinking of surface IgM produces an intracellular calcium flux inIgM⁺ CD22⁺ but not in IgM⁺ CD22⁻ cells (Pezzutto et al., J. Immunol.140:1791-1795 (1988)). These results suggest that, like T cell accessorymolecules, CD22 may also participate in the regulation of signaltransduction.

The ability to interfere with the binding of CD22 positive B cells withaccessory cells, or the ability to cause such binding to occur onsurfaces other than lymphocyte cells can be useful in diagnostics andtherapy. For example, a fusion protein of CD22 and a receptor ligandfixed to a substrate will be useful in detecting the presence of aparticular antigen in body fluids. A soluble form of CD22 can haveimmunomodulatory activity.

EXAMPLE XIII The Isolation and Molecular Cloning of cDNA Encoding for TLymphocyte-specific CD27 Antigen

A cDNA clone encoding CD27 was obtained from human T lymphocyte cDNAtransferred into COS cells and immunoselected by the method of thepresent invention. RNA was extracted from the mononuclear cells derivedfrom a unit of blood, after four days of culture in medium containing 1ug/ml phytohemagglutinin (PHA), using guanidium thiocyanate. The totalRNA was poly-A selected. cDNA was made and cloned into CDM8, transfectedinto COS cells and the CD27 cDNA was immunoselected with monoclonalantibodies OKT18a and CLB-9F4 (provided as described in Seed and AruffoProc. Natl. Acad. Sci. 84:8573-8577 (1987); and Aruffo and Seed Proc.Natl. Acad. Sci. USA 84:3365-3369 (1987)). The vector contained a 1.2 kbcDNA insert.

The nucleotide sequence of the cDNA was determined by dideoxynucleotidechain termination as described, supra. The sequence of 1203 residues andthe deduced amino acid sequence appear in Table 5. The initiationmethionine is indicated by the number 1 above the initiator codon. Thededuced CD27 polypeptide demonstrates the typical features of a type Iintegral membrane protein. It begins with a twenty amino acidhydrophobic region consistent with a secretory signal sequence. Thishydrophobic region is followed by a 171 residue extracellular domain, a20 residue hydrophobic membrane spanning domain (doubly underlined) anda 49 amino acid cytoplasmic domain beginning with a positively chargedstop transfer sequence. There is no poly(A) tail.

The deduced CD27 amino acid sequence is highly homologous to the Blymphocyte and carcinoma antigen CD40, described supra, over its entirelength. CD27 is also highly homologous to the the the receptor for nervegrowth factor (NGFR) over the extracellular and transmembrane domains(Stamenkovic et al., EMBO J. 8:1403-1410 (1989); Johnson et al., Cell47:545-554 (1989)). The most conserved structural motif found in thesethree proteins is the abundance of cysteines or histidines in theextracellular region. These are often found in pairs separated by two orfour intervening amino acid residues similar to the arrangement seen inproteins which use this structure to bind a zinc ion. The cysteine andhistidine rich region is followed by a serine, threonine and prolinerich membrane proximal domain which has been suggested to be the regionin which biochemically identified O-linked glycans are added to NGFR(Johnson et al. (1986), supra; Grob et al., J. Biol. Chem. 260:8044-8049(1985)).

                                      TABLE 5                                     __________________________________________________________________________    1GGGGTGCAAAGAAGAGACAGCAGCGCCCAGCTTGGAGGTGCTAACTCCAGAGGCCAGCATCAGCAACTGGGCA     ##STR69##                                                                     ##STR70##                                                                     ##STR71##                                                                     ##STR72##                                                                     ##STR73##                                                                     ##STR74##                                                                     ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                     ##STR78##                                                                     ##STR79##                                                                    961TGGCAGCCACAACTGCAGTCCCATCCTCTTGTCAGGGCCCTTTCCTGTGTACACGTGACAGAGTGCCTTTT    G                                                                             1041CAGGGACGAGGACAAATATGGATGAGGTGGAGAGTGGGAAGCAGGAGCCCAGCCAGCTGCGCGCGCGTGC    G                                                                             1121GGGCTCTGGTTGTAAGGCACACTTCCTGCTGCGAAAGACCCACATGCTACAAGACGGGCAAAATAAAGTG    __________________________________________________________________________

Immunoprecipitation of transfected COS cells with anti-CD27 antibodiesfollowed by gel electrophoresis revealed the presence of a 110 kdspecies when not reduced and a single 55 kd band in the presence ofreducing agent. This indicates that on transfected COS cells, CD27 is adisulfide linked homodimer comprised of 55 kd monomers, similar to theforms precipitated from T lymphocytes. (Bigler et al., J. Immunol.141:21-28 (1988); Stockinger et al., Leukocyte Typing II, Vol. I:513-529(1986); Van Lier et al., Eur. J. Immunol. 18:811-816 (1987)).

CD27 is a T lymphocyte activation antigen. Its structure suggests thatit may function as the receptor for a lymphokine or growth factor. Therecognition of CD27 causes T cell proliferation and increased expressionof certain genes needed for the helper and effector functions of the Tcell. The expression of CD27 on T cells increases two to five fold withstimulation by phytohemagglutinin (PHA) or anti-CD3 monoclonalantibodies and the addition of at least one CD27 monoclonal antibody canaugment PHA stimulated proliferation of T cells (Bigler and Chiorazzi,Leukocyte Typing II, Vol. I:503-512 (1986); Van Lier, (1987)). T cellspositive for CD27 have been found to provide help to B cells for IgMsynthesis and secrete Il-2 when appropriately stimulated (Van Lier etal., Eur. J. Immunol. 18:811-816 (1988)).

The ability to interfere with the binding of CD27 positive T cells withantigen presenting cells, or the ability to cause such binding to occuron surfaces other than lymphocyte cells, can be useful in diagnosticsand therapy. For example, a fusion protein of CD27 and a receptor ligandfixed to a substrate will be useful in detecting the presence of aparticular antigen in body fluids. A soluble CD27 fusion protein will beuseful to prevent undesired T cell proliferation, for example, incertain autoimmune diseases.

EXAMPLE XIV The Isolation and Molecular Cloning of the Two cDNA ClonesEncoding T Lymphocyte-specific Leu8 Antigens

Two cDNA clones encoding Leu8 determinants were isolated from a human Tcell library by the method of the present invention.

The nucleotide sequence of the cDNA was determined by dideoxynucleotidechain termination as described, supra. The DNA sequence analyses (Table6) shows that the longer insert of the two contains 2,350 residues,whereas the shorter lacks 436 internal residues but is otherwiseidentical. The entire sequence of the longer clone is shown, with theportion deleted from the shorter clone overlined. The predicted aminoacid sequence is shown below the nucleotide sequence. Sites of potentialN-linked glycosylation are designated --CHO-- and the proposedtransmembrane region for the longer form is doubly underlined.

                                      TABLE 6                                     __________________________________________________________________________     ##STR80##                                                                     ##STR81##                                                                     ##STR82##                                                                     ##STR83##                                                                     ##STR84##                                                                     ##STR85##                                                                     ##STR86##                                                                     ##STR87##                                                                     ##STR88##                                                                     ##STR89##                                                                     ##STR90##                                                                     ##STR91##                                                                     ##STR92##                                                                     ##STR93##                                                                     ##STR94##                                                                     ##STR95##                                                                     ##STR96##                                                                     ##STR97##                                                                     ##STR98##                                                                     ##STR99##                                                                    1601AATATGGACTCAGTTTTCTTGCAGATCAAATTTCACGTCGTCTTCTGTATACTGTGGAGGTACACTCTTA    1681AAAAAGTCTACGCTCTCCTTTCTTTCTAACTCCAGTGAAGTAATGGGGTCCTGCTCAAGTTGAAAGAGTC    T                                                                             1761TGTAGCCTCGCCGTCTGTGAATTGGACCATCCTATTTAACTGGCTTCAGCCTCCCCACCTTCTTCAGCCA    C                                                                             1841TCAGTTGGCTGACTTCCACACCTAGCATCTCATGAGTGCCAAGCAAAAGGAGAGAAGAGAGAAATAGCCT    C                                                                             1921TTAGTTTGGGGGTTTTGCTGTTTCCTTTTATGAGACCCATTCCTATTTCTTATAGTCAATGTTTCTTTTA    C                                                                             2001ATTAGTAAGAAAACATCACTGAAATGCTAGCTGCAAGTGACATCTCTTTGATGTCATATGGAAGAGTTAA    A                                                                             2081GAAATTCCTTGATTCACAATGAAATGCTCTCCTTTCCCCTGCCCCCAGACCTTTTATCCGACTTACCTAG    T                                                                             2161TTCTTTAAATTTCATCTCAGGCCTCCCTCAACCCCACCACTTCTTTTATAACTAGTCCTTTACTAATCCA    C                                                                             2241AGCTCCTCTTCCTGGCTTCTTACTGAAAGGTTACCCTGTAACATGCAATTTTGCATTTGAATAAAGCCTG    T                                                                             2321GTTAAAAAAAAAAAAAAAAAAAAAAAAAAA                                            __________________________________________________________________________

DNA blot hybridization of fragmented human T cell genome showed apattern consistent with a single copy gene. RNA blot hybridizationrevealed a major transcript of 2.4 kb in peripheral blood mononuclearcells, tonsillar B cells, and several lymphocytic cell lines; and aminor transcript of 2.0 kb, present in peripheral blood mononuclearcells, and the Jurkat and HSB-2 leukaemic T cell lines.

The deduced protein encoded by the larger insert (the conventional form)bears a strongly hydrophobic putative membrane spanning domain near itsC terminus, followed by

several positively charged residues resembling a cytoplasmic anchorsequence. The protein is closely related to the recently describedmurine Mel-14 homing receptor (Lasky et al., Cell 56:1045-1055 (1989);Siegelman et al., Science 243:1165-1172 (1989)).

The protein encoded by the shorter insert (the phospholipid anchoredform) bears a weakly hydrophobic C-terminal domain characteristic ofsurface proteins that are attached to the cell membrane by covalentlinkage to a phosphatidylinositol-substituted glycan.

Monoclonal antibodies TQ1 (Reinherz et al. (1982) J. Immun. 128:463-468)and Mel-14 (Gallatin et al. (1983) Nature 304:30-34) have been observedto react with COS cells transfected with either Leu8 clone.

The presence or absence of Leu8 on CD4+ T lymphocytes identifiessuppressor-inducer and helper-inducer CD4+ T cell subsets. Leu8 is ahoming receptor, allowing T cells to adhere to the specializedpost-capillary endothelium of peripheral lymph nodes. The presence orabsence of Leu8 classifies the T cell in terms of homing potential andtissue distribution. Serological studies have indicated that Leu8 is amarker of resting lymphocytes in peripheral lymph nodes (Poletti et al.,Hum. Pathol. 19:1001-1007 (1988)). Activation of T cells by phorbolester plus PHA results in reduced Leu8 expression and transcripts, withthe reduction in Leu8 expression being more rapid than the reduction ofLeu8 transcripts. It therefore appears that surface Leu8 is lost morerapidly than predicted by RNA turnover, possibly by shedding of thephosphatidylinositol-linked form (Ferguson & Williams, Ann. Rev.Biochem. 57:285-320 (1988)). Among peripheral T cells, the CD4⁺ Leu8⁻subset provides help for B cell IgM and IgG synthesis (Reinherz et al.,J. Immun. 128:463-468 (1982); Gatenby et al., J. Immun. 129:1997-2000),whereas CD4⁺ Leu8⁻ cells have been found to directly inhibit pokeweedmitogen-induced IgG synthesis (Kanof et al., J. Immun. 139:49-54(1987)). It therefore appears that CD4⁺ Leu8⁻ cells, activated toprovide help for B cell Ig synthesis, exit the nodes and circulateperipherally to encounter antigen-presenting cells.

The ability to interfere with the binding of Leu8⁻ T cells to antigenpresenting cells, or the ability to cause such binding to occur onsurfaces other than lymphocyte cells, can be useful in diagnostics andtherapy. For example, the level of activated Leu8⁻ T cells relative toresting Leu8⁺ cells could serve as a measure of immune response to aparticular antigen.

The extracellular domain of the Leu8 transmembrane protein, whichmediates adhesion to specialized endothelial cells of lymph nodes, hasbeen observed to be quite specific in its recognition of the lectinligand, sulfated galactosyl ceramide (sulfatide). Modification of thespecificity of this binding could serve to regulate the homing potentialof resting T cells. Soluble forms of Leu8 can act as anti-inflammatoryagents by reducing lymphocyte migration.

EXAMPLE XV The Isolation and Molecular Cloning of cDNAs Encoding CD44Antigens

CD44 is a polymorphic integral membrane protein. Immunochemical and RNAblot data have supported the existence of two forms of CD44: amesenchymal form expressed by hematopoietic cells and an epithelial formweakly expressed by normal epithelium but highly expressed bycarcinomas.

To isolate a cDNA clone encoding hematopoietic CD44 (Stamenkovic et al.,Cell 56:1057-1062 (1989)), libraries prepared from the histiocyticlymphoma cell line U937, the B lymphoblastoid line JY, the Burkitt'slymphoma line Raji, and the myeloid leukemia line KG-1 were transfectedseparately into COS cells by the DEAE-Dextran method, described supra.The cells were pooled 48 hours after transfection, incubated withanti-CD44 monoclonal antibodies J173 (Pesandro et al., J. Immunol.137:3689-3695 (1986)), and panned on dishes coated with goat antimouseaffinity purified antibody. After several washes, the adherent cellswere lysed, and episomal DNA was purified and transformed into E. coli.After two similar rounds of enrichment following spheroplast fusion,plasmid DNA recovered from three out of eight randomly picked colonieswas found to direct the appearance of hematopoietic CD44 determinants ontransfected COS cells.

Two of the three clones, CD44.5 and CD44.8, bore inserts of about 1.4kb, while the third, CD44.4, contained an insert of about 1.7 kb. COScells transfected with either of these clones reacted with anti-CD44monoclonal antibodies J173, F-10-44-2 (Dalchau et al., Eur. J. Immunol.10:745-749 (1980)) and the anti-Pgp-1 monoclonal antibody IM7(Trowbridge et al., Immunogenetics 15:299-312 (1982)). Untransfectedcells showed weak J173 reactivity.

The nucleotide sequence of the hematopoietic CD44.5 cDNA (Table 7)consists of 1354 residues terminating in a short poly(A) tail 19 basepairs downstream from a CATAAA sequence. The ATG encoding the firstmethionine is embedded in a consensus initiation sequence and followedby 19 predominantly hydrophobic residues resembling a secretory signalpeptide sequence. Cleavage of this peptide would yield a mature proteinof 341 residues with a predicted relative molecular mass of 37.2 kd. Theextracellular amino terminal domain of 248 residues is followed by 21predominantly hydrophobic amino acids corresponding to the predictedtransmembrane domain (doubly underlined) and a 72 residue hydrophilic(cytoplasmic) domain. The discrepancy between the predicted mass of theprotein backbone and the deglycosylated forms observed inimmunoprecipitates suggest that extensive O-linked glycosylation ispresent. The extracellular domain has six potential N-linkedglycosylation sites, indicated in Table 7 by a --CHO-- designation, andis rich in serine and threonine residues (22% in aggregate). Thedipeptide SG that forms the minimal attachment site of serine-linkedchondroitin sulfate in proteoglycan proteins appears at residues 160,170, 211 and 238 in the predicted extracellular domain; these potentialglycosylation sites are underlined.

    TABLE 7                                                                          -                                                                              1CCAGCCTCTGCCAGGTTCGGTCCGCCATCCTCGTCCCGTCCTCCGCCGGCCCCTGCCCCGCGCCCAGGGATC    C                                                                                ##STR100##                                                                     ##STR101##                                                                     ##STR102##                                                                     ##STR103##                                                                     ##STR104##                                                                     ##STR105##                                                                     ##STR106##                                                                     ##STR107##                                                                     ##STR108##                                                                     ##STR109##                                                                     ##STR110##                                                                     ##STR111##                                                                     ##STR112##                                                                     ##STR113##                                                                                                                                             1     201ACACCTACACCATTATCTTGGAAAGAAACAACCGTTGTAAACATAACCATTACAGGGAGCTGGGACACTTA    A                                                                                                                                                        1     281ATGTGCTACTGATTGTTTCATTGCGAATCTTTTTTAGCATAAAATTTTCTACTCTTTTTGTTAAAAAAAAA    A                                                                               354                                                                       

RNA blot hybridization revealed three major messages of 1.6, 2.2 and 5.0kb in a variety of hematopoietic cell lines, including the Blymphoblastoid line CESS, the T cell leukemias HUT-102 and HPB-ALL,lymphokine activated T cells, tonsillar B cells and the histiocyticlymphoma U937.

Immunoprecipitation of CD44 from transfected COS cells reveals that themesenchymal or hematopoietic form of CD44 is about 80-90 kd.Hematopoietic CD44 transfected into a B cell line has been observed toresult in the binding of the CD44-bearing lymphocytes to rat lymph nodestromal cells in primary culture, indicating that hematopoietic CD44 mayplay a role in lymphocyte homing. It has been shown that hematopoieticCD44 is an extracellular matrix receptor with affinity for collagenstype I and VI (Stamenkovic et al., Cell 56:1057-1062 (1989)).Hematopoietic CD44 may also have a lymphocyte activation role.

The ability to interfere with the binding of hematopoietic CD44 to lymphnode cells, or the ability to cause such binding to occur on othersurfaces, can be useful in diagnostics and therapy. For example,modification of this binding can serve to regulate the homing potentialof lymphocytes. Soluble forms of CD44 can have immunomodulatoryactivity.

To isolate a cDNA clone encoding the epithelial form of CD44, a cDNAlibrary prepared from the colon carcinoma line HT29 was transfected intoCOS cells by the DEAE-Dextran method described supra. The cells werepooled 48 hours after transfection, incubated with anti-CD44 monoclonalantibody F-10-44-2 (Dalchau et al., Eur. J. Immunol. 10:745-749 (1980))and panned on dishes coated with goat-anti-mouse affinity purifiedantibody. After several washes, the adherent cells were lysed, andepisomal DNA purified and transformed into E. coli. After two similarrounds of enrichment following spheroplast fusion, as described supra,plasmid DNA recovered from seven out of ten randomly picked colonies wasfound to direct the appearance of epithelial CD44 determinants ontransfected COS cells. All seven of the positive clones bore cDNAinserts of about 2.4 kb.

Restriction enzyme analysis of the clone containing the epithelial cDNAinsert showed that the coding sequence (Table 8) was enlarged relativeto the hematopoietic CD44 insert by the addition of 496 base pair. DNAsequence analysis showed that the epithelial CD44 cDNA is quite similarto the CD44.5 cDNA, but encoded an additional extracellular domain of165 amino acids, inserted about 140 residues upstream of thetransmembrane section shared by both clones. The mature protein wouldcomprise 493 residues.

RNA blot analysis has revealed that the epithelial CD44 transcriptscomprise 2.2, 2.7 and 5.5 kb species. Epithelial CD44 isolated byimmunoprecipitation has revealed that the glycoprotein is about 160 kd.

    TABLE 8                                                                          -                                                                              1CCAGCCTCTGCCAGGTTCGGTCCGCCATCCTCGTCCCGTCCTCCGCCGGCCCCTGCCCCGCGCCCAGGGATC    C                                                                                ##STR114##                                                                     ##STR115##                                                                     ##STR116##                                                                     ##STR117##                                                                     ##STR118##                                                                     ##STR119##                                                                     ##STR120##                                                                     ##STR121##                                                                     ##STR122##                                                                     ##STR123##                                                                     ##STR124##                                                                     ##STR125##                                                                     ##STR126##                                                                     ##STR127##                                                                     ##STR128##                                                                     ##STR129##                                                                     ##STR130##                                                                     ##STR131##                                                                     ##STR132##                                                                                                                                             1     601CTACACCATTATCTTGGAAAGAAACAACGTTGGAAACATAACCATTACAGGGGAGCTGGGACACTTAACAG    A                                                                                                                                                        1     681GCTACTGATTGTTTCATTTCGAATCTATAATAGCATAAAATTTTCTACTCTTTTTGTTTTTTGTGTTTTGT    T                                                                                                                                                        1     761TCAGGTCCAATTTGTAAAAACAGCATTGCTTTCTGAAATTAGGGCCCAATTAATAATCAGCAAGAATTTTG    A                                                                                                                                                        1     841GTTCCCCACTTGGAGGCCTTTCATCCCTCGGGTGTGCTATGGATGGCTTCTAACAAAAACCTACCACATAG    T                                                                                                                                                        1     921ATCGCCAACCTTGCCCCCCACCAGCTAAGGACATTTCCAGGGTTAATAGGGCCTGGTCCTGGGAGGAAATT    T                                                                                                                                                        2     001CATTTTGCCCTTCCATTAGCCTAATCCCTGGGCATTGCTTTCCACTGAGGTTGGGGGTTGGGGTGTACTAG    T                                                                                                                                                        2     081TTCAACAGACCCCCTCTAGAAATTTTTCAGATGCTTCTGGGAGACACCCAAAGGGTAAGTCTATTTATCTG    T                                                                                                                                                        2     161ATTTATCTGTGTTTTTGAAATATTAAACCCTGGATCAGTCCTTTTATTCAGTATAATTTTTTAAAGTTACT    T                                                                                                                                                        2     241GCACAAAAAGGGTTTAAACTGATTCATAATAAATATCTGTACCTTCTTCGAAAAAAAAAAAAAAAAAA   

Transfected B cells expressing epithelial CD44 do not adhere to ratlymph node stromal cells in primary culture as do hematopoietic CD44transfected lymphocytes. The epithelial CD44 is weakly expressed bynormal epithelium but highly expressed by carcinomas. It is possiblethat an extracellular matrix receptor function of epithelial CD44 maypromote tumor invasiveness.

The ability to interfere with the binding of epithelial CD44 withextracellular matrices can be useful in therapy or diagnostics. Forexample, interference of the epithelial CD44 binding to extracellularmatrices can diminish the likelihood of metastasis in cancer patients.Soluble forms of CD44 can act to prevent metastatic cells from "homing"to lymph nodes.

EXAMPLE XVI The Isolation and Molecular Cloning of cDNA Encoding CD53Antigens

CD53, the antigen recognized by antibodies MEM-53 (Hadam, M. R. (1989)in Leucocyte Typing IV, Knapp, B. et al. (eds.) Oxford Univ. Press, p.674), HD77, H129 and HI36, and 63-5A3, is a glycoprotein widelydistributed among, but strictly restricted to, nucleated cells of thehematopoietic lineages (Stevanova, I., et al., (1989) in LeucocyteTyping IV, Knapp, B. et al. (eds.) Oxford Univ. Press, p. 678). CD53 isexpressed by monocytes and macrophages, by granulogytes, dendriticcells, osteoclasts and osteoblasts, and by T and B cells from everystage of differentiation.

To obtain a cDNA clone encoding CD53, cDNA libraries constructed fromperipheral blood lymphocytes and the promyelocytic tumor cell line HL60were transfected into COS cells by the DEAE-Dextran method, describedsupra. The cells were pooled 48 hours after transfection, incubated withmonoclonal antibodies MEM-53, and panned as described in Seed andAruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987) and Aruffo andSeed, Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987). After twosubsequent rounds of enrichment following spheroplast fusion, plasmidDNA recovered from single colony isolates was transfected into COScells, and scored for CD53 expression by immunofluorescence.

Two of eight transfectants were positive, each bearing an insert ofabout 1.5 kb. COS cells transfected with either clone reacted with eachof the antibodies MEM-53, HI29, HI36 and 63-5A3.

To isolate CD53 from peripheral blood lymphocytes and transfected COScells for purposes of comparison, the lymphocytes and transfected COScells were surface labeled with ¹²⁵ I using lactoperoxidase and H₂ O₂,and then lysed in a lysis buffer of 50 mM Tris-HCl pH 8.0 containing 1%NP40, 150 mM NaCl, 5 mM MgCl₂, 5 mM KCl, 20 mM iodoacetamide and 1 mMphenylmethylsulfonyl-fluoride. Cells were solubilized at a concentrationof 2.5×10⁷ cells/ml in lysis buffer for 45 minutes then centrifuged at12,000 g. After preclearing with goat anti-mouse immunoglobulin beads(Cappel, Malvern, Pa.), immunoprecipitations were performed withmonoclonal antibodies MEM53 or 63-5A3 and protein A-Sepharose CL-4B(Sigma, St. Louis, Mo.) as described by Schneider et al. (J. Biol. Chem.257:10766 (1982)). Immunoprecipitates were eluted in SDS-sample bufferand analyzed on 12.5% acrylamide gels containing sodium dodecyl sulfate.

A broad band of radioiodinated protein ranging in mass from 34 kd to 42kd was obtained from peripheral blood lymphocytes either with MEM-53 or63-5A3 monoclonal antibodies, comparable to the band obtained fromCD53-transfected COS cells, which extended from 36 kd to 46 kd. Thehigher molecular mass in COS cells is atypical, as cell surface proteinsrecovered from transfected COS cells usually display unchanged or lowermolecular mass than those found on the cell from which the cDNA cloneoriginated (Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84:3365-3369(1987)). Approximately 15 kd of mass are liberated from the glycoproteinby digestion with endoglycosidase F (N-glycanase), whereas treatmentwith neuraminidase and O-glycanase has no effect on the apparentmolecular mass (Hadam, M. R. (1989) in Leucocyte Typing IV, Knapp, B. etal. (eds.) Oxford Univ. Press, p. 674; Stevanova et al., in LeucocyteTyping IV, Knapp, B. et al. (eds.) Oxford Univ. Press, p. 678). Anadditional faint band of 20 kd, possibly unglycosylated precursor, wasdetected in immunoprecipitates of transfected COS cells, but was absentfrom immunoprecipitates of peripheral blood lymphocytes.

Blot hybridization of genomic DNA from the T cell line PEER digestedwith several enzymes revealed a pattern consistent with a single copygene. RNA blot analysis revealed a single 1.8 kb mRNA derived from B, T,and myeloid cell lines and from peripheral blood lymphocytes. The levelof expression was comparable in the different cell lines except in THP1cell line, which had little CD53 mRNA. CD53 transcripts are moreabundant in peripheral blood lymphocytes than in cultivated cell lines,consistent with the higher surface expression of CD53 among these cells.

The nucleotide sequence of CD53 cDNA was determined by dideoxynucleotidechain termination as described, supra, using synthetic oligonucleotideprimers. The sequence of the CD53 insert consists of 1452 nucleotides(Table 9), and terminates close to two overlapping AATAAA motifs (singlyunderlined). The 3' noncoding sequence contains three examples of theATTTA sequence (indicated by quotation marks), which has been shown tomediate mRNA instability (Shaw and Kamen, Cell 46:659 (1986)).

    TABLE 9                                                                          -                                                                              ##STR133##                                                                     ##STR134##                                                                     ##STR135##                                                                     ##STR136##                                                                     ##STR137##                                                                     ##STR138##                                                                     ##STR139##                                                                     ##STR140##                                                                     ##STR141##                                                                     ##STR142##                                                                                                                                             8     01AGCCCTACATGATCACTGCAGGATGATCCTCCTCCCATCCTTTCCCTTTTTAGGTCCCTGTCTTATACAACC    A                                                                                                                                                        8     81GGGTGTTGGCCAGGCACATCCCATCTCAGGCAGCAAGACAATCTTTCACTCACTGACGGCAGCAGCCATGTC    T                                                                                ##STR143##                                                                                                                                             1     041AACCCAGGATATGAATTTTTGCATCTTCCCATTGTCGAATTAGTCTCCAGCCTCTAAATAATGCCCAGTCT    T                                                                                                                                                        1     121AGTCAAGCAAGAGACTAGTTGAAGGGAGTTCTGGGGCCAGGCTCACTGGACCATTGTCACAACCCTCTGTT    T                                                                                                                                                        1     201CTAAGTGCCCTGGCTACAGGAATTACACAGTTCTCTTTCTCCAAAGGGCAAGATCTCATTTCAATTTCTTT    A                                                                                ##STR144##                                                                     ##STR145##                                                                     ##STR146##                                                               

An open reading frame beginning at residue 74 encodes a protein of 219amino acids with a predicted molecular weight of 24,340 kd. Thepredicted polypeptide is unusual in that it bears four major hydrophobicsegments (doubly underlined), three of which fall in close proximitynear the amino terminus of the molecule. The first hydrophobic segmentis atypically long for either a signal sequence or a simpletransmembrane alpha helix, and contains three cysteine residues and aglycine located in the middle. Both cysteine and glycine have been foundto immediately precede the signal cleavage site (von Heijne, NucleicAcids Res. 14:4683 (1986)), suggesting that the amino terminus of themature protein begins in the middle of the first hydrophobic domain.Some support for this view is afforded by the finding that the size ofthe polypeptide backbone of ME491, a related type III integral membraneprotein, discussed infra, is smaller than the size predicted from thecDNA sequence, possibly as a consequence of signal peptide excision.Because there are only two potential sites for N-linked glycan addition(indicated in Table 9 by --CHO-- designations), located between thethird and fourth hydrophobic segments, the carboxyl terminus must lieinside the cell, as well as the short hydrophilic portion between thesecond and third hydrophobic segments. If the amino terminus is notprocessed, it must likewise remain intracellular.

CD53 is a Type III integral membrane protein related to three othermembrane proteins: ME491 antigen, a melanoma protein whose increasedexpression correlates well with tumor progression (Hotta et al., CancerRes. 48:2955 (1988)); CD37, an extensively glycosylated antigenpredominantly expressed on B cells, but not B cell lineage specific(Schwartz et al., J. Immun. 140:905 (1988); and S5.7, an unglycosylatedantigen broadly expressed on cells of hematopoietic lineage (Pressano etal., Cancer Res. 43:4812 (1983)). In addition, CD53 is distantly relatedto E. coli lac Y permease, a type III integral membrane protein whichferries lactose into the bacterial cell. CD53 transcripts in peripheralblood lymphocytes increase in prevalence following mitogenic stimulationby PHA, suggesting that the protein may be involved in the transport offactors essential for cell proliferation.

Among the molecules with broad reactivity in the hemopoietic system,CD53 presently holds the widest reactivity as well as the strictestrestriction to hematopoietic cells. Anti-CD53 antibodies are a usefultool for the identification of hematopoietic neoplasms, and may provehelpful for identifying morphologically poorly defined cells, forexample in spleen or bone marrow primary cultures.

Equivalents

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed within the scope of this invention.

We claim:
 1. A cloned cDNA encoding a human CD40 cell surface antigen ora functional derivative of said cDNA encoding at least 6 amino acids ofhuman CD40.
 2. Substantially pure cDNA comprising the nucleotidesequence shown in FIG. 16 or a functional derivative of at least 18nucleotides thereof, wherein said derivative encodes at least 6 aminoacids of human CD40.